Articles de revues sur le sujet « "nor tomato mutant" »

Abstract Differences in aroma have been examined in headspace samples of ripe tomato (Lycopersicon esculentum Mill.) fruit of ‘Rutgers’ and of yellowing fruit of the nonripening mutants rin and nor. Volatiles were trapped and separated by gas chromatography, and the intensity of the effluent aromas was rated by sniffing. Intense aroma compounds were identified by mass spectrometry. Sixty-nine intense compounds were found in ‘Rutgers’, of which 46 were present in one or both mutant strains. Fifteen compounds with odor intensities rated medium to very strong were identified that were deficient or absent in fruit of the mutants. The latter compounds included two aldehydes, seven alcohols, two ketones, three sulfur-containing compounds, and a phenol. A few compounds were intense odors in ‘Rutgers’ and in one or both mutants; hex-2-enal, linalool, phenylacetaldehyde, methyl salicylate, 2-phenylethanol, and eugenol. Some compounds were detected that were more intense in rin and nor than in ‘Rutgers’ (e.g., guaiacol). It is proposed that the “normal background aroma” in fresh tomatoes is caused by those intense odors, which are common to both normal and mutant strains, whereas the bland flavor of mutant fruit is caused by the absence of those intense aroma compounds found only in ‘Rutgers’. The intense aroma compounds found only in ‘Rutgers’ may be crucial determinants of acceptability in fresh tomato fruit.

Abstract The tomato non-ripening (nor) mutant generates a truncated 186-amino-acid protein (NOR186) and has been demonstrated previously to be a gain-of-function mutant. Here, we provide more evidence to support this view and answer the open question of whether the NAC-NOR gene is important in fruit ripening. Overexpression of NAC-NOR in the nor mutant did not restore the full ripening phenotype. Further analysis showed that the truncated NOR186 protein is located in the nucleus and binds to but does not activate the promoters of 1-aminocyclopropane-1-carboxylic acid synthase2 (SlACS2), geranylgeranyl diphosphate synthase2 (SlGgpps2), and pectate lyase (SlPL), which are involved in ethylene biosynthesis, carotenoid accumulation, and fruit softening, respectively. The activation of the promoters by the wild-type NOR protein can be inhibited by the mutant NOR186 protein. On the other hand, ethylene synthesis, carotenoid accumulation, and fruit softening were significantly inhibited in CR-NOR (CRISPR/Cas9-edited NAC-NOR) fruit compared with the wild-type, but much less severely affected than in the nor mutant, while they were accelerated in OE-NOR (overexpressed NAC-NOR) fruit. These data further indicated that nor is a gain-of-function mutation and NAC-NOR plays a significant role in ripening of wild-type fruit.

Abstract Fungal cell-wall lysing enzymes have been shown to induce ethylene production in different plant systems. The effect of endogenous plant cell-wall lysing enzymes on ethylene synthesis in fruit has received only limited attention. Therefore, tomato fruit (Lycopersicon esculentum, Mill.) were vacuum–infiltrated with the tomato cell-wall enzymes, polygalacturonase I and II (PG I, PG II) and pectinmethylesterase (PME). Fruit ethylene levels were observed to increase relative to either salt, buffer, or boiled enzyme controls. This increase in ethylene production occurred in green ‘Cherry’ tomato fruit as well as in the mutants rin, nor, and Cornell 111. Enzyme-induced ethylene synthesis generally peaked at or before 17 to 20 hr and decreased to lower or basal levels in most immature normal cultivars by 42 hr after treatment. Ethylene was maintained at high levels, however, in some (possibly more mature) green fruit, as well as in all mutant lines. PG II was more effective than PG I in inducing ethylene production and PME seemed to enhance the ethylene-inducing activity of PG II.

The insertion site of a transposon mutant of Clavibacter michiganensis subsp. michiganensis NCPPB382 was cloned and found to be located in the gene tomA encoding a member of the glycosyl hydrolase family 10. The intact gene was obtained from a cosmid library of C. michiganensis subsp. michiganensis. The deduced protein TomA (543 amino acids, 58 kDa) contains a predicted signal peptide and two domains, the N-terminal catalytic domain and a C-terminal fibronectin III-like domain. The closest well-characterized relatives of TomA were tomatinases from fungi involved in the detoxification of the tomato saponin α-tomatine which acts as a growth inhibitor. Growth inhibition of C. michiganensis subsp. michiganensis by α-tomatine was stronger in the tomA mutants than in the wild type. Tomatinase activity assayed by deglycosylation of α-tomatine to tomatidine was demonstrated in concentrated culture supernatants of C. michiganensis subsp. michiganensis. No activity was found with the tomA mutants. However, neither the transposon mutant nor a second mutant constructed by gene disruption was affected in virulence on the tomato cv. Moneymaker.

Recent advances in molecular genetics and genomics technologies have had a significant impact on tomato research over the last decade and are likely to have considerable influence on the nature and outcome of research activities related to tomato in the future. Specific applications of genomics technologies in our laboratory include positional cloning of genes associated with fruit ripening and quality (rin and nor), localization of ripening-related genes on the molecular-marker map to assist candidate gene discovery related to fruit ripening and quality, and characterization of mutants influencing fruit quality and nutritional value with the goal of identifying candidate genes for said mutants and alternative molecular tools for modification of fruit quality and nutrition. Isolation of the rin and nor genes has been verified via complementation of corresponding mutant tomato plants via insertion of the appropriate CaMV35s-driven wild-type sense cDNA. Both the rin and nor genes have sequence characteristics suggestive of transcription factors. Preliminary evidence suggests the role of similar genes in the ripening of additional climacteric and non-climacteric fruit species. Additional efforts in the laboratory include molecular analyses of light signal transduction as related to 1) regulation of carotenoid and flavonoid accumulation, and 2) potential manipulation of corresponding pathways for modification of fruit quality and nutrient value.

Abstract: The objective of this work was to evaluate the effects of the mutant alleles alcobaça (nor A ), ripening inhibitor (rin), and old gold crimson (og c ), in heterozygosity or homozygosity, on the expression of color and on the postharvest quality of fruit of experimental tomato hybrids. Fourteen hybrids with contrasting genotypic constitutions in the nor A , rin, and og c loci were evaluated in a randomized complete block design with four replicates. The following fruit postharvest quality traits were evaluated: firmness in the breaker stage, color, and soluble solids content. The rin +/rin and nor +/nor A genotypes increased firmness of tomato fruit at harvest (breaker stage). The rin + /rin genotypes displayed the worse internal fruit color. There was a positive effect of og c+ /og c in improving the internal color of rin + /rin and nor + /nor A fruit, making the color similar to that of the normal genotypes. The combination of the og c /og c rin + /rin nor + /nor A genes is effective to improve tomato fruit firmness, besides maintaining or improving internal color.

Two tomato lines with normal maturation (NR-1 and NR-2) have been crossed with two mutant lines (NR-10 nor and NR-12 rin) with delayed maturation (shelf life). Determination of mutant genes has been done by ?2 test on 100 fruits from F2 generation. Fruits have been picked 65 days from antesis and kept for 60 days, when six evaluations have been done. Data have been collected every 10 days on parental lines and progeny F1 and F2 generation. Variance testing has been done on the basis of one- and two-factorial analysis and groups compared by contrasts. Fruits have been preserved in controlled conditions (in dark at 5?C). Tomato genotypes with nor or rin gene had desirable traits (delayed ripening, long shelf life and firm fruits) for modern selection, so they should be included in programmes aiming to create commercial F1 hybrids.

Bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, continues to be a problem for tomato growers worldwide. A collection of nonpathogenic bacteria from tomato leaves plus P. syringae strains TLP2 and Cit7, P. fluorescens strain A506, and P. syringae pv. tomato DC3000 hrp mutants were examined in a greenhouse bioassay for the ability to reduce foliar bacterial speck disease severity. While several of these strains significantly reduced disease severity, P. syringae Cit7 was the most effective, providing a mean level of disease reduction of 78% under greenhouse conditions. The P. syringae pv. tomato DC3000 hrpA, hrpH, and hrpS mutants also significantly reduced speck severity under greenhouse conditions. The strains with the greatest efficacy under greenhouse conditions were tested for the ability to reduce bacterial speck under field conditions at locations in Alabama, Florida, and Ontario, Canada. P. syringae Cit7 was the most effective strain, providing a mean level of disease reduction of 28% over 10 different field experiments. P. fluorescens A506, which is commercially available as Blight-Ban A506, provided a mean level of disease reduction of 18% over nine different field experiments. While neither P. syringae Cit7 nor P. fluorescens A506 can be integrated with copper bactericides due to their copper sensitivity, there exist some potential for integrating these biological control agents with “plant activators”, including Actigard. Of the P. syringae pv. tomato DC3000 hrp mutants tested, only the hrpS mutant reduced speck severity significantly under field conditions.

Responses of resistant (Mi-1/Mi-1) and susceptible (mi-1/ mi-1) tomato (Solanum lycopersicum) to root-knot nematodes (RKNs; Meloidogyne spp.) infection were monitored using cDNA microarrays, and the roles of salicylic acid (SA) and jasmonic acid (JA) defense signaling were evaluated in these interactions. Array analysis was used to compare transcript profiles in incompatible and compatible interactions of tomato roots 24 h after RKN infestation. The jai1 and def1 tomato mutant, altered in JA signaling, and tomato transgenic line NahG, altered in SA signaling, in the presence or absence of the RKN resistance gene Mi-1, were evaluated. The array analysis identified 1,497 and 750 genes differentially regulated in the incompatible and compatible interactions, respectively. Of the differentially regulated genes, 37% were specific to the incompatible interactions. NahG affected neither Mi-1 resistance nor basal defenses to RKNs. However, jai1 reduced tomato susceptibility to RKNs while not affecting Mi-1 resistance. In contrast, the def1 mutant did not affect RKN susceptibility. These results indicate that JA-dependent signaling does not play a role in Mi-1-mediated defense; however, an intact JA signaling pathway is required for tomato susceptibility to RKNs. In addition, low levels of SA might be sufficient for basal and Mi-1 resistance to RKNs.

The tomato is a research model for fruit-ripening, however, its fruit-ripening mechanism still needs more extensive and in-depth exploration. Here, using TMT and LC-MS, the proteome and phosphoproteome of AC++ (wild type) and rin (ripening-inhibitor) mutant fruits were studied to investigate the translation and post-translational regulation mechanisms of tomato fruit-ripening. A total of 6141 proteins and 4011 phosphorylation sites contained quantitative information. One-hundred proteins were identified in both omics’ profiles, which were mainly found in ethylene biosynthesis and signal transduction, photosynthesis regulation, carotenoid and flavonoid biosynthesis, chlorophyll degradation, ribosomal subunit expression changes, MAPK pathway, transcription factors and kinases. The affected protein levels were correlated with their corresponding gene transcript levels, such as NAC-NOR, MADS-RIN, IMA, TAGL1, MADS-MC and TDR4. Changes in the phosphorylation levels of NAC-NOR and IMA were involved in the regulation of tomato fruit-ripening. Although photosynthesis was inhibited, there were diverse primary and secondary metabolic pathways, such as glycolysis, fatty acid metabolism, vitamin metabolism and isoprenoid biosynthesis, regulated by phosphorylation. These data constitute a map of protein—protein phosphorylation in the regulation of tomato fruit-ripening, which lays the foundation for future in-depth study of the sophisticated molecular mechanisms of fruit-ripening and provide guidance for molecular breeding.

Sinaloa tomato growers continuously evaluate new varieties, looking for better quality and long shelf life. Mutant fruit with the genes rin and nor offers both possibilities when crossed with normal fruit. Our study presents results of 16 tomato hybrid lines harvested from the field at the “turning” stage and stored under simulated marketing conditions (20°C and 80% RH). Twelve experimental hybrids were from the rin type, one from the nor type, two commercial hybrids were normal, and one commercial line from the rin type. Sampling was done every 2 days up to 16 days. Evaluations done included physical, chemical, and physiological determinations. Line S69 (nor type) had higher firmness compared to the others, while normal lines (S121 and S123) were the softest. S69 was the only hybrid that did not completed a red external color development. Experimental lines of the rin type presented acceptable development of red color, however, only normal lines (S121 and S123) reached the characteristic red color of tomato. Lines of the rin type (S172 and S200) lost more weight during marketing than normal ones. Pulp pH was higher on the experimental rin lines than on the commercial ones (BR84, S121 and S123). Not difference on the sugar: acid ratio among the lines was found. Only normal lines showed a climateric CO2 and C2H4 peak.

Abstract Postchilling ion leakage, respiration, and C2H4 biosynthesis were used to measure the degree of chilling injury to fruit of tomato (Lycopersicon esculentum Mill. ‘Heinz 1350’). Chilling sensitivity, as measured by ion leakage, first declined as the tomatoes began to ripen and then increased during the late stages of ripening. Both C2H4 biosynthesis and respiration rate were stimulated during the chilling response early in ripening. In the nonripening mutant, nor, chilling sensitivity did not show the early decline but showed the increase during senescence. Variation in chilling sensitivity during tomato ripening was biphasic, with a decline at the onset of ripening followed by a senescence-related increase. It is proposed that the latter may be due to an increase in membrane viscosity.

Root colonization of Arabidopsis thaliana by the nonpathogenic, rhizosphere-colonizing, biocontrol bacterium Pseudomonas fluorescens WCS417r has been shown to elicit induced systemic resistance (ISR) against Pseudomonas syringae pv. tomato (Pst). The ISR response differs from the pathogen-inducible systemic acquired resistance (SAR) response in that ISR is independent of salicylic acid and not associated with pathogenesis-related proteins. Several ethylene-response mutants were tested and showed essentially normal symptoms of Pst infection. ISR was abolished in the ethylene-insensitive mutant etr1-1, whereas SAR was unaffected. Similar results were obtained with the ethylene-insensitive mutants ein2 through ein7, indicating that the expression of ISR requires the complete signal-transduction pathway of ethylene known so far. The induction of ISR by WCS417r was not accompanied by increased ethylene production in roots or leaves, nor by increases in the expression of the genes encoding the ethylene biosynthetic enzymes 1-aminocyclopropane-1-carboxylic (ACC) synthase and ACC oxidase. The eir1 mutant, displaying ethylene insensitivity in the roots only, did not express ISR upon application of WCS417r to the roots, but did exhibit ISR when the inducing bacteria were infiltrated into the leaves. These results demonstrate that, for the induction of ISR, ethylene responsiveness is required at the site of application of inducing rhizobacteria.

The root-knot nematode (RKN) is one of the most devastating parasitic nematodes of plants. Although some secondary metabolites released by the host plant play roles as defense substances against parasitic nematodes, the mechanism underlying the induction of such defense responses is not fully understood. We found that sclareol, a natural diterpene known as an antimicrobial and defense-related molecule, inhibited RKN penetration of tomato and Arabidopsis roots. Sclareol induced genes related to ethylene (ET) biosynthesis and signaling and phenylpropanoid metabolism in Arabidopsis roots. In roots of ein2-1, an ET-insensitive mutant line, both sclareol-induced inhibition of RKN penetration and sclareol-induced enhancement of lignin accumulation were abolished. A mutant defective in lignin accumulation did not exhibit such inhibition. Sclareol also activated MPK3 and MPK6, Arabidopsis mitogen-activated protein kinases whose activation is required for triggering ET biosynthesis. Sclareol-induced inhibition of RKN penetration was exhibited by mutants of neither MPK3 nor MPK6. Treatment with a biosynthetic precursor of ET was insufficient compared with sclareol treatment to inhibit RKN penetration, suggesting the existence of an ET-independent signaling pathway leading to RKN resistance. These results suggested that sclareol induced resistance to RKN penetration partially through ET-dependent accumulation of lignin in roots.

ABSTRACT The host-specific plant pathogen Pseudomonas syringaeelicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the P. syringae genome. hrpW was found in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42.9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from Nectria haematococca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity inA 230 assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the P. syringae pv. syringae B728a hrpW was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the P. syringae pv. tomato hrpW were found in other P. syringaepathovars, Pseudomonas viridiflava, Ralstonia(Pseudomonas) solanacearum, andXanthomonas campestris. ΔhrpZ::nptII orhrpW::ΩSpr P. syringaepv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in ahrpZ hrpW double mutant. These features of hrpWand its product suggest that P. syringae produces multiple harpins and that the target of these proteins is in the plant cell wall.

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicislycopersici. The production of phenazine-1-carboxamide (PCN) is crucial for this biocontrol activity. In vitro production of PCN is observed only at high-population densities, suggesting that production is under the regulation of quorum sensing. The main autoinducer molecule produced by PCL1391 was identified structurally as N-hexanoyl-l-homoserine lactone (C6-HSL). The two other autoinducers that were produced comigrate with N-butanoyl-l-homoserine lactone (C4-HSL) and N-octanoyl-l-homoserine lactone (C8-HSL). Two PCL1391 mutants lacking production of PCN were defective in the genes phzI and phzR, respectively, the nucleotide sequences of which were determined completely. Production of PCN by the phzI mutant could be complemented by the addition of exogenous synthetic C6-HSL, but not by C4-HSL, C8-HSL, or any other HSL tested. Expression analyses of Tn5luxAB reporter strains of phzI, phzR, and the phz biosynthetic operon clearly showed that phzI expression and PCN production is regulated by C6-HSL in a population density-dependent manner. The introduction of multiple copies of the regulatory genes phzI and phzR on various plasmids resulted in an increase of the production of HSLs, expression of the PCN biosynthetic operon, and consequently, PCN production, up to a sixfold increase in a copy-dependent manner. Surprisingly, our expression studies show that an additional, yet unidentified factor(s), which are neither PCN nor C4-HSL or C8-HSL, secreted into the growth medium of the overnight cultures, is involved in the positive regulation of phzI, and is able to induce PCN biosynthesis at low cell densities in a growing culture, resulting in an increase of PCN production.

Nitric oxide (NO) has been suggested to play a role in the hypersensitive response (HR). Single- and double-label fluorescence microscopy experiments were conducted using Arabidopsis leaves infected with Pseudomonas syringae pv. tomato DC3000 carrying either avrB or avrRpt2. Kinetics of NO production were followed by measurement of green 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) triazole fluorescence in leaves coinfiltrated with DAF-FM diacetate. Kinetics of hypersensitive cell death were followed by measurement of cytoplasmic red fluorescence following internalization of coinfiltrated propidium iodide through compromised plasma membranes. Neither NO accumulation nor cell death was seen until approximately 3 h postinoculation of Columbia leaves with DC3000·avrB or approximately 5.5 h post-inoculation with DC3000·avrRpt2. Subsequent NO accumulation kinetics closely paralleled HR progression in both Columbia and ndr1-1 mutant plants. These data established that NO accumulation does not happen sufficiently early for NO to be a signaling component controlling HR triggering. NO accumulation did contribute to the HR, as proven by an approximately 1-h delay in cell death kinetics caused by an NO scavenger or an NO synthase inhibitor. NO was first seen as punctate foci at the cell surface. Subsequent NO accumulation patterns were consistent with NO being an intercellular signal that functions in cell-to-cell spread of the HR.

ABSTRACT When coresident with the Ti (tumor-inducing) plasmid, the 21-kDa product of the osa gene of the plasmid pSa can suppress crown gall tumorigenesis incited by Agrobacterium tumefaciens. Neither T-DNA processing nor vir(virulence) gene induction is affected by the presence ofosa in the bacterium. We used Arabidopsis thaliana root segments and tobacco leaf discs to demonstrate that Osa inhibits A. tumefaciens from transforming these plants to the stable phenotypes of tumorigenesis, kanamycin resistance, and stable β-glucuronidase (GUS) expression. When A. tumefaciens contained osa, the lack of expression of transient GUS activity in infected plant tissues, as well as the lack of systemic viral symptoms following agroinfection of Nicotiana benthamiana by tomato mottle virus, suggested that oncogenic suppression by Osa occurs before T-DNA enters the plant nucleus. The extracellular complementation of an A. tumefaciens virE2mutant (the T-DNA donor strain) by an A. tumefaciens strain lacking T-DNA but containing a wild-type virE2 gene (the VirE2 donor strain) was blocked when osa was present in the VirE2 donor strain, but not when osa was present in the T-DNA donor strain. These data indicate that osa inhibits VirE2 protein, but not T-DNA export from A. tumefaciens. These data further suggest that VirE2 protein and T-DNA are separately exported from the bacterium. The successful infection of Datura stramonium plants and leaf discs of transgenic tobacco plants expressing VirE2 protein by an A. tumefaciens virE2 mutant carrying osa confirmed that oncogenic suppression byosa does not occur by blocking T-DNA transfer. Overexpression of virB9, virB10, andvirB11 in A. tumefaciens did not overcome oncogenic suppression by osa. The finding that the expression of the osa gene by itself, rather than the formation of a conjugal intermediate with pSa, blocks transformation suggests that the mechanism of oncogenic suppression by osamay differ from that of the IncQ plasmid RSF1010.

Cucumber mosaic virus (CMV) associated with D satellite RNA (satRNA) causes lethal systemic necrosis (LSN) in tomato (Solanum lycopersicum), which involves programmed cell death. No resistance to this disease has been found in tomato. We obtained a line of wild tomato, S. habrochaitis, with a homogeneous non-lethal response (NLR) to the infection. This line of S. habrochaitis was crossed with tomato to generate F1 plants that survived the infection with NLR, indicating that NLR is a dominant trait. The NLR trait was successfully passed on to the next generation. The phenotype and genotype segregation was analyzed in the first backcross population. The analyses indicate that the NLR trait is determined by quantitative trait loci (QTL). Major QTL associated with the NLR trait were mapped to chromosomes 5 and 12. Results from Northern blot and in situ hybridization analyses revealed that the F1 and S. habrochaitis plants accumulated minus-strand satRNA more slowly than tomato, and fewer vascular cells were infected. In addition, D satRNA-induced LSN in tomato is correlated with higher accumulation of the minus-strand satRNA compared with the accumulation of the minus strand of a non-necrogenic mutant D satRNA.

5'-methylthioadenosine (MTA) nucleosidase (EC.2.2.2.28) and 5-methylthioribose (MTR) kinase (EC.2.7.1.100) activities were evaluated in `rin', `nor', and `Rutgers' tomato fruit during development and ripening. Changes in the activities of these enzymes were compared to ethylene biosynthesis. MTA nucleosidase and MTR kinase activities in `rin' and `nor' were ≈30% and 22%, respectively, lower than `Rutgers' during the first 2 weeks of fruit development. In `Rutgers', activities of these enzymes declined sharply until fruit maturity. Shortly before climacteric rise in ethylene synthesis, MTA nucleosidase, and MTR kinase activities increased, reaching a maximum level before peak ethylene synthesis then declined when fruit started to approach senescence. Whereas, `rin' and `nor' mutants exhibited no climacteric rise in ethylene synthesis and no change in MTA nucleosidase or MTR kinase activities, following their decline after 2 weeks of growth. A rapid increase in ethylene synthesis was observed when mature green `rin' and `nor' fruit were wounded. This increase in ethylene was paralleled by an increase in MTA nucleosidase and MTR kinase activities. However, increase in wound ethylene, MTA nucleosidase, and MTR kinase activities in `rin' and `nor' was ≈40% less than what we had previously reported in `Rutgers'. Relationship of MTA and MTR kinase activities to fruit growth, development, ripening, and natural and wound ethylene biosynthesis will be described.

In this work the bio-availability of amino acids for the root-colonizing Pseudomonas fluorescens strain WCS365 in the tomato rhizosphere was studied. The amino acid composition of axenically collected tomato root exudate was determined. The results show that aspartic acid, glutamic acid, isoleucine, leucine, and lysine are the major amino acid components. The concentrations of individual amino acids in the rhizosphere of gnotobiotically grown tomato plants were estimated and considered to be too low to support growth of rhizosphere micro-organisms to numbers usually found in the tomato rhizosphere. To test this experimentally, mutants of P. fluorescens WCS365 auxotrophic for the amino acids leucine, arginine, histidine, isoleucine plus valine, and tryptophan were isolated after mutagenesis with Tn5lacZ. Root tip colonization of these mutants was measured after inoculation of germinated tomato seeds and subsequent growth in a gnotobiotic quartz sand system (M. Simons, A. J. van der Bij, I. Brand, L. A. de Weger, C. A. Wijffelman, and B. J. J. Lugtenberg. 1996. Gnotobiotic system for studying rhizo-sphere colonization by plant growth-promoting Pseudomonas bacteria. Mol. Plant-Microbe Interact. 9:600–607). In contrast to the wild-type strain, none of the five amino acid auxotrophs tested was able to colonize the tomato root tip, neither alone nor after co-inoculation with the wild-type strain. However, addition of the appropriate amino acid to the system restored colonization by the auxotrophic mutants, usually to wild-type levels. Analysis of the root base showed that cells of auxotrophic mutants were still present there. The results show that, although amino acids are present in root exudate, the bio-availability of the tested amino acids is too low to support root tip colonization by auxotrophic mutants of P. fluorescens strain WCS365. The genes that are required for amino acid synthesis are therefore necessary for root colonization. Moreover, these compounds apparently play no major role as nutrients in the tomato rhizosphere.

Plants and animals rely on immune receptors, known as nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins, to defend against invading pathogens and activate immune responses. How NLR receptors respond to pathogens is inadequately understood. We previously reported single-residue mutations that expand the response of the potato immune receptor R3a to AVR3aEM, a stealthy effector from the late blight oomycete pathogen Phytophthora infestans. I2, another NLR that mediates resistance to the wilt-causing fungus Fusarium oxysporum f. sp. lycopersici, is the tomato ortholog of R3a. We transferred previously identified R3a mutations to I2 to assess the degree to which the resulting I2 mutants have an altered response. We discovered that wild-type I2 protein responds weakly to AVR3a. One mutant in the N-terminal coiled-coil domain, I2I141N, appeared sensitized and displayed markedly increased response to AVR3a. Remarkably, I2I141N conferred partial resistance to P. infestans. Further, I2I141N has an expanded response spectrum to F. oxysporum f. sp. lycopersici effectors compared with the wild-type I2 protein. Our results suggest that synthetic immune receptors can be engineered to confer resistance to phylogenetically divergent pathogens and indicate that knowledge gathered for one NLR could be exploited to improve NLR from other plant species.

Abstract A vacuum infiltration technique that allowed precise control of both infiltration rate and amount of solution administered to whole tomato (Lycopersicon esculentum) fruit was developed. Controlled volumes of 5 mm solutions of CuSO4, Cu(NO3)2, HgCl2, CaSO4, KNO3, (NH4)2C2O4, Na2HPO4, citrate and 1 mm EDTA or EGTA were infiltrated into intact, mature-green tomato fruit and evaluated with regard to their effect on the pattern of tomato ripening. Copper significantly accelerated lycopene accumulation and influenced both the timing and magnitude of climacteric ethylene production. Infiltration with HgCl2 elicited similar effects as copper, but severe phytotoxicity was observed. In contrast, CaSO4, KNO3, and chelators had no significant effect on the pattern of ripening. Copper initiated wound ethylene production in the ripening mutant rin that reached up to 50% of the wound levels observed in normal fruit, but rin was not induced to ripen.

Glycine-rich RNA-binding proteins (GRPs) are involved in the modulation of the post-transcriptional processing of transcripts and participate as an output signal of the circadian clock. However, neither GRPs nor the circadian rhythmic have been studied in detail in fleshy fruits as yet. In the present work, the GRP1 gene family was analysed in Micro-Tom tomato (Solanum lycopersicum L.) fruit. Three highly homologous LeGRP1 genes (LeGRP1a–c) were identified. For each gene, three products were found, corresponding to the unspliced precursor mRNA (pre-mRNA), the mature mRNA and the alternatively spliced mRNA (preLeGRP1a–c, mLeGRP1a–c and asLeGRP1a–c, respectively). Tomato GRPs (LeGRPs) show the classic RNA recognition motif and glycine-rich region, and were found in the nucleus and in the cytosol of tomato fruit. By using different Escherichia coli mutants, it was found that LeGRP1s contained in vivo RNA-melting abilities and were able to complement the cold-sensitive phenotype of BX04 cells. Particular circadian profiles of expression, dependent on the fruits’ developmental stage, were found for each LeGRP1 form. During ripening off the vine of fruits harvested at the mature green stage, the levels of all LeGRP1a–c forms drastically increased; however, incubation at 4°C prevented such increases. Analysis of the expression of all LeGRP1a–c forms suggests a positive regulation of expression in tomato fruit. Overall, the results obtained in this work reveal a complex pattern of expression of GRPs in tomato fruit, suggesting they might be involved in post-transcriptional modulation of circadian processes of this fleshy fruit.

Bacillus cereus AR156 (AR156) is a plant growth–promoting rhizobacterium capable of inducing systemic resistance to Pseudomonas syringae pv. tomato in Arabidopsis thaliana. Here, we show that, when applied to Arabidopsis leaves, AR156 acted similarly to flg22, a typical pathogen-associated molecular pattern (PAMP), in initiating PAMP-triggered immunity (PTI). AR156-elicited PTI responses included phosphorylation of MPK3 and MPK6, induction of the expression of defense-related genes PR1, FRK1, WRKY22, and WRKY29, production of reactive oxygen species, and callose deposition. Pretreatment with AR156 still significantly reduced P. syringae pv. tomato multiplication and disease severity in NahG transgenic plants and mutants sid2-2, jar1, etr1, ein2, npr1, and fls2. This suggests that AR156-induced PTI responses require neither salicylic acid, jasmonic acid, and ethylene signaling nor flagella receptor kinase FLS2, the receptor of flg22. On the other hand, AR156 and flg22 acted in concert to differentially regulate a number of AGO1-bound microRNAs that function to mediate PTI. A full-genome transcriptional profiling analysis indicated that AR156 and flg22 activated similar transcriptional programs, coregulating the expression of 117 genes; their concerted regulation of 16 genes was confirmed by real-time quantitative polymerase chain reaction analysis. These results suggest that AR156 activates basal defense responses to P. syringae pv. tomato in Arabidopsis, similarly to flg22.

Relevance. High lycopene fruit content has been regarded as a very important genetic trait in tomato breeding. Use lycopene molecular markers in combination with conventional breeding techniques allowed us to create hybrids with high lycopene accumulation, excellent organoleptic qualities, high yield production and resistance to pathogens, and to effectively optimize our breeding programmes for commercial greehouses production.Material and Methods. In this study tomato samples including selected lines and hybrids with various allelic combinations of genes determining carotene accumulation, and other genetic traits, such as disease resistance and yield production were tested. Introgression of spontaneous and induced mutations was used to increase carotenoid levels (og and hp) and improve fruit technological qualities (nor, alc, rin). The research material was tomato collection, mutants, breeding lines and hybrids listed in the State Register Russian Federation tomato hybrids of breeding SS Agrofirm "Ilyinichna" VNIIO branch of the All-Russian Scientific Research Institute of Vegetable Growing – Branch of the FSBSI Federal Scientific Vegetable Center. DNA typing of fruit quality genes was performed at the Institute of Genetics and Cytology of the National Academy of Sciences of Belarus.Results. New domestic hybrids for industrial greenhouses, which characterised by improved organoleptic qualities and technological traits were developed with the help of phasedcross-breeding that allowed to combine the genes nor, rin, alc, leading to an extension of the shelf life with the genes B, og, hp1, etc., contributing to an increase in carotenoid content in fruits. It was established that for targeted selection and hybridization, despite the negative influence of the nor, rin, alc genes it is possible to raise the level of carotenoids to average values. Correlation between lycopene concentration in fruits and high temperature and level of insolation was confirmed. It was shown that pink-fruited forms contain significantly more lycopenethanred-fruitedones. Different all eliccombinations of structural genes involved in carotenoids biosynthesis and regulatory genes that provided maximal accumulation of lycopene in hybrid swithred and pink fruits were revealed. Hybrids with the combination of high concentrations of sugar (° Brix), dry matter and maximal lycopene values, combined defining excellent taste were selected: Prekrasnaiya lady, Olya, Quadrille, Victoria. New F1 hybrids one for industrial greenhouses: G950, G956, G960, Magistral and pink fruited G12897, surpassed the Dutch standard in productivity up to 21%, and in tastes/organoleptic qualities for 1-1.8 points.

Following perception of a pathogenic attack, plants are able to develop a strong response with the corresponding activation of a plethora of defense-related genes. In this study we have characterized the mode of expression of the CEVI-1 gene from tomato plants, which encodes an anionic peroxidase. CEVI-1 expression is induced during the course of compatible viral and subviral infections, like many other defense-related genes, but is induced neither in incompatible interactions nor by signal molecules such as salicylic acid, ethylene, or methyl jasmonate. Additionally, CEVI-1 is induced in detached leaf tissues following a pathway distinct from that related to the classical wound response. We also describe the characterization of the structural CEVI-1 gene and compare the mode of expression in different transgenic plant species harboring a CEVI-1::GUS construct. Furthermore, we have isolated mutants in Arabidopsis, called dth mutants, that are deregulated in the control of expression of this gene. From the initial analysis of some of these mutants it seems that activation of CEVI-1 gene expression correlates with a defect in the perception of auxins by the plant. All these results may suggest that, during systemic infections with viruses, auxin homeostasis is one of the components participating in the regulation of the overall defense response.

Abstract Tomato (Lycopersicon esculentum Mill. cv. Heinz 1350) plants grown in soil with N supplied from (NH4)2SO4 solutions showed a morphological disorder characterized by leaf epinasty. The development of this disorder was accompanied by an increase in the rate of ethylene evolution from whole plants. Ethylene evolution from plants supplied with 0.04 m NH4-N increased to a peak of 112 nl·g−1·hr−1 at ≈2 weeks following the start of fertilization compared to 11 nl·g−1·hr−1 from plants supplied with 0.04 m NO3-N. Fertilization with KC1 in molar equivalency to the supply of NH4-N prevented epinasty and the burst in ethylene evolution. Ethylene evolution from plants of the yellow-green-5 and neglecta-1 mutants did not increase in response to NH4-N fertilization. Potassium concentrations in shoots of ‘Heinz 1350% yellow-green- 5, and neglecta-1 were 2.10, 2.53, and 3.22% (dry weight), respectively, if plants were supplied with NH4-N and no additional K, suggesting that tolerance to NH4 toxicity may be explained in part by differences in K accumulation.

Abstract Many plant species open their leaves during the daytime and close them at night as if sleeping. This leaf movement is known as nyctinasty, a unique and intriguing phenomenon that been of great interest to scientists for centuries. Nyctinastic leaf movement occurs widely in leguminous plants, and is generated by a specialized motor organ, the pulvinus. Although a key determinant of pulvinus development, PETIOLULE-LIKE PULVINUS (PLP), has been identified, the molecular genetic basis for pulvinus function is largely unknown. Here, through an analysis of knockout mutants in barrelclover (Medicago truncatula), we showed that neither altering brassinosteroid (BR) content nor blocking BR signal perception affected pulvinus determination. However, BR homeostasis did influence nyctinastic leaf movement. BR activity in the pulvinus is regulated by a BR-inactivating gene PHYB ACTIVATION TAGGED SUPPRESSOR1 (BAS1), which is directly activated by PLP. A comparative analysis between M. truncatula and the non-pulvinus forming species Arabidopsis and tomato (Solanum lycopersicum) revealed that PLP may act as a factor that associates with unknown regulators in pulvinus determination in M. truncatula. Apart from exposing the involvement of BR in the functionality of the pulvinus, these results have provided insights into whether gene functions among species are general or specialized.

ABSTRACT The nucleus-localized C2 protein of Tomato yellow leaf curl virus-China (TYLCV-C) is an active suppressor of posttranscriptional gene silencing (PTGS). Consistently, infection with TYLCV-C resulted in PTGS arrest in plants. The C2 protein possesses a functional, arginine-rich nuclear localization signal within the basic amino acid-rich region 17KVQHRIAKKTTRRRR31. When expressed from potato virus X, C2-RRRR31DVGG (in which the four consecutive arginine residues 28RRRR31 were replaced with DVGG) that had been tagged with a green fluorescent protein (GFP) failed to transport GFP into nuclei and was dysfunctional in inducing necrosis and suppressing PTGS in plants. Amino acid substitution mutants C2-K17D-GFP, C2-HR21DV-GFP, and C2-KK25DI-GFP localized to nuclei and produced necrosis, but only C2-K17D-GFP suppressed PTGS. The N-terminal portions C21-31 and C217-31 fused in frame to GFP were capable of targeting GFP to nuclei, but neither caused necrosis nor affected PTGS. Our data establish that nuclear localization is likely required for C2 protein to function in C2-mediated induction of necrosis and suppression of PTGS, which may follow diverse pathways in plants. Possible mechanisms of how the C2 protein involves these biological functions are discussed.

Nitrogen is a paramount important essential element for all living organisms. It has been found to bea crucial structural component of proteins, nucleic acids, enzymes and other cellular constituents which are inevitable for all forms of life. In the atmosphere, the percentage of nitrogen is very high (N2, 78%) compared to other inorganic gases. However, most organisms have practically no direct access to this nitrogen. While plants can not directly uptake nitrogen from atmosphere, they are capable of assimilating other forms of nitrogen, for example ammonium (NH4+) and nitrate (NO3-). For agricultural crop production, artificial fixation of nitrogen is heavily utilized and it is an expensive process that requires high temperatures (at least 400 °C) and pressures (around 200 atm). It has been conspicuously demonstrated that indiscriminate use of fertilizer hampers soil physical, chemical and micro biological properties and also a potential risk to environment e.g. water quality. Besides, chemically manufactured fertilizers are depleted from soils in various ways, for instance; denitrifying bacteria, volatilization, and leaching. Consequently, it results relatively poor availability of nitrogen to get into plants. On the flipside, only 1-2% of the nitrogen fixation in the world occurs through the natural process of lightening. Notably, microbial fixation is well characterized in diazotrophs for example; Rhizobia and Frankia, and blue-green algae. Against the backdrop, we are accentuated on an environmentally friendlyand themost sustainable approach to increase productivity for legume and non-legume crops. Till today, the term biological nitrogen fixation (BNF) has received much attention as a sustainable alternative; this process facilitates atmospheric nitrogen to convert into ammonia by rhizobia in specialized plan organs termed “root nodules”. This review article seeks to better understand plant mechanisms involved in the development of root nodules in soybean. Soybean (Glycine max) is one of the most important oil crops and a source of animal feed protein in the world. It has a salient feature to fix atmospheric nitrogen through symbioses with compatible rhizobia that yields to determinate type nodule (Oldroyd, Murray et al. 2011). Biological nitrogen fixation in soybean nodules reduces the use of chemical nitrogen fertilizers resulting in cost-savings to producers and minimizes environmental damage due to nitrogen run-off. A better understanding of how nodules form and function is important for selection or generation of soybean genotypes with better nitrogen fixation capacity. Soybean nodules originate from root cortex via de novo cell differentiation (Oldroyd 2013). Consequently, two major nodule development zones are formed for instance; the nodule primordium (Npr) in the middle and it is encircled by nodule parenchyma (Npa). At later time point, the Npr gives rise to N-fixation zone and the Npa holds vascular bundles. It is not clear what early signaling pathways driving the conspicuous development of the nodule zones. My research is aimed at filling this knowledge gap by illustrating the molecular signatures that paves the way to cellular differentiation in root nodule development in soybean. Based on initial evidence obtained by the Subramanian lab, we hypothesize that microRNAs (miRNAs) play important regulatory roles in spatio-temporal expression of their target genes during nodule developmental in soybean. For instance, the regulation of auxin sensitivity by miR160 has been found to be crucial for formation of nodule primordia and vasculature in the parenchyma (Marie Turner 2013). Against this backdrop, this review article focused on nuclear and cytoplasmic transcriptome as well as miRNA profiles of parenchyma and primordial tissues and determine the relative abundance and differentially expressed mRNAs and regulatory role of miRNAs in cell differentiation and nodule development. Root nodule a sustainable alternative to fix atmospheric nitrogen Atmospheric nitrogen percentage is very high (N2, 78%) compared to other inorganic gases (Mary Elvira 1932). However, most of the organisms have practically no direct access to this nitrogen. Nevertheless, plants can not directly uptake nitrogen from atmosphere but they are capable of assimilating only very specific forms of nitrogen, for example ammonium (NH4+) and nitrate (NO3-) (Bytnerowicz and Fenn 1996, Peter M. Vitousek 1997) (Sponseller, Gundale et al. 2016). Virtually, nitrogen has been found to be a crucial structural component of proteins, nucleic acids, enzymes, and other cellular constituents which are inevitable for all forms of life (O'Brien, Vega et al. 2016). For agricultural crop production, artificial fixation of nitrogen is heavily utilized. It is an expensive process that requires high temperatures (approx. 400 °C) and pressures (approx. 200 atm) (Witschi 2000). It has been conspicuously demonstrated that indiscriminate use of N fertilizer hampers the diversity of the bacterial community and decreases soil C and N concentrations (Verzeaux, Alahmad et al. 2016). Notably, it has been demonstrated as a potential risk to environment e.g. water quality (Zhao, Sha et al. 2016) (Sponseller, Gundale et al. 2016). Besides, chemically manufactured fertilizers are depleted from soils in various ways, for instance; denitrifying bacteria, volatilization, and leaching (Johnson 1996, Peter M. Vitousek 1997). Consequently, it results relatively poor availability of nitrogen to get into plants. On the flipside, over 90 % of the nitrogen fixation in the world occurs through the natural process of lightening and microorganisms. Furthermore, microbial fixation is well characterized in diazotrophs for example; Rhizobia and Frankia, and blue-green algae (Cheng 2008). It has been demonstrated that Bradyrhizobium strains substantially escalated soybean grain yield, and protein content up to 57% and 26%, respectively (Zimmer, Messmer et al. 2016). Against the backdrop, we are accentuated on an environmentally friendly and a sustainable approach to increase the productivity for legume and non-legume crops. Literature mining depicted that biological nitrogen fixation in soybean nodules reduces the use of chemical nitrogen fertilizers resulting in cost-savings to producers and minimizes environmental damage due to nitrogen run-off. Rhizobia infection leads to the root nodule development In the natural environment, plants are continuously confronted with pathogenic and symbiotic microbes. Symbioses involves mutual exchange of diffusible signal molecules, first endophytic bacteria (rhizobia) are attracted by the plant root exudates flavonoids which are perceived and triggered the bacterial nodulation (nod) genes. Consequently, the bacteria synthesize specific lipochito-oligosaccharides, called nodulation (Nod) factors. This signal is perceived by the LysM receptor like kinase of host plant, it induces the root hair curling, and bacteria get access into the host epidermis through infection threads (ITs) and initiate cell division within the root cortex, leading to the progression of the root nodule meristem. In later stages of the interaction, bacteria are released from the infection threads into the plant cells, surrounded by membrane of plant origin. These bacteria multiply within the host cells and differentiate into the nitrogen fixing bacteroids (Udvardi and Day 1997) (Oldroyd 2013). Till now, integration of genetic and genomic approaches has revealed twenty-six genes to be involved in nodule development of Medicago truncatualaand Lotus japonicum (Kouchi, Imaizumi-Anraku et al. 2010). In addition, deep sequencing of the Medicago truncatularoot transcriptome has uncovered thousands of genes to be induced during Nod factor signaling and its resulting ethylene (ET) biosynthesis throughout the multiple development stages of indeterminate nodule (Larrainzar, Riely et al. 2015). Albeit the molecular mechanism of such regulation is not well understood. There has been a large-scale transcriptome analysis of B. japonicum-inoculated and mock-inoculated soybean root hairs. It has showed that a total of 1,973 soybean genes differentially expressed during root hair infection, particularly NFR5 and NIN genes (Libault, Farmer et al. 2010). Nevertheless, the signaling mechanisms directing the cellular differentiation of nodule are not known. Soybean root nodule organogenesis Soybean (Glycine max) has a genome size of 1.1 to1.5 Gb, it is partially diploidized tetraploid. It is one of the most important oil crops and a source of animal feed protein in the world (soybase.org/sb_about.php). It has a salient feature to fix atmospheric nitrogen through symbioses with compatible rhizobia that yields to determinate type nodule (Udvardi and Day 1997) (Oldroyd, Murray et al. 2011). Notwithstanding of the economic and environmental importance, there has been very few studies about quantitative trait loci (QTL) that controlling BNF traits, for instance nodule number, ration of nodule dry weight with nodule number, and shoot dry weight (SDW). It has been reported via composite interval mapping that approximately six QTLs bears very small effect on BNF traits (Santos, Geraldi et al. 2013). Besides, it has been demonstrated in earlier studies that nodules originate from root cortex via de novo cell differentiation into two different cell types, parenchymal and primordium (Celine Charon 1997) (Oldroyd&Downie 2008; Oldroyd 2013). In addition, early nodulin genes in legume for instance; Enod 40 gene reported to be expressed in root pericycle during the rhizobia infection and later it occupied in the dividing cortical cells (H. Kouchi and S. Hata 1993). Among the two major nodule development zones, the nodule primordium (Npr) in the middle which is encircled by nodule parenchyma (Npa). At later time point, the Npr gives rise to N-fixation zone and the Npa holds vascular bundles. Lately, a β- expansin gene, GmEXPB2 fused with GUS reporter gene which was observed to be preferentially expressed in nodule vascular trace and nodule vascular bundles. It indicated that GmEXPB2 might be crucial for nodule organogenesis. Over expression of GmEXPB2 contrast to suppressed GmEXPB2 transgenic lines found to be escalated nodule number, nodule mass and nitrogenase activity. It further suggested that GmEXPB2 might have influenced over root architecture, nodule formation and development, and profoundly yielding to biological N2 fixation (Li, Zhao et al. 2015). Even though, it is not clear what early signaling pathways driving the conspicuous development of the nodule zones. Against the back drop, to understand the regulation of auxin sensitivity by miR160 which is believed to be crucial for the formation of nodule primordia (Marie Turner 2013). Figure 1 a. Illustrating the progression of root nodule development through Rhizobial bacterial infection in the plant root leading to the determinate nodule (Oldroyd 2013). b. Nodule development zones A. Nodule primordial zone (Enod 40 gene) in the middle B. surrounding parenchyma (Enod 2 gene), differentiated from cortex (collected from Sen Subramanian lab). Regulatory small RNAs biogenesis and its molecular functions Regulatory small RNAs are ranged between 20 to 24 nucleotides which are ubiquitous elements of endogenous plant transcriptomics, a common response to exogenous viral infections and introduced double-stranded RNA (Axtell 2013). Three core enzymes families, for instance; RNAdependent RNA polymerase (RDR), Dicer like (DCL), and Argonaute (AGO) proteins paves the way of small RNA biogenesis and function in plants. Firstly, ribonuclease type III or DICERLIKE1 involves in the yield of a fold-back precursor RNA or primary miRNA (primiRNA) transcripts using an RNA templates in the nuclei. Later, the resulting miRNA-miRNA duplex which is originated in nucleus then translocated into cytoplasm. The guided miRNAmolecule is incorporated into ARGONAUTE (AGO) to form an active RISC complex to specific target RNAs that are complementary to the miRNA, and this process eventually follows up mRNA cleavage, represses the translation of the mRNAs or Chromatin modification. This phenomenon accentuated as an inhibition or silencing of the gene expression, which play a crucial role in the developmental process in plant and animal (Chapman and Carrington 2007) (Axtell 2013). Fig. 2 Regulation of gene expression events via RISC complex (modified from https://www.google.com/?gws_rd=ssl#q=mirna+picture+in+plants accessed on 7th February, 2016) Fig. 3 Gene expression events occurring in typical plant cell (modified https://www.google.com/search?q=transcription+and+translation accessed on 7th February, 2016) It has been found in several studies that most plant miRNAs are non-coding RNA, and small 21-24 nucleotide long (Cuperus, Fahlgren et al. 2011). It requires DCL1-clade DCL for their biogenesis and AGO1-clade AGO for their function (Wu, Zhou et al. 2010, Manavella, Koenig et al. 2012). In rice (Oryza sativa), DCL3 has been reported in the biogenesis of 24nt long miRNA that incorporated in AGO4 to regulate the target gene expression primarily through mRNA cleavage (Wu, Zhou et al. 2010). Argonaute proteins (AGO) form RNA inducing silencing complexes (RISC) with small RNAswhich is known as post-transcriptional gene silencing. It has typically four domains, for instance:N-terminal, PAZ, MID and PIWI domains. The MID-PIWI lobes are belongs to the C-terminus. It has been studied that MID-domains contains the specificity loop to recognize and bind to the 5’-phosphate of smRNAs. The PIWI domains contained the catalytic active site D-E-D-H/D. PAZ domain anchored the 2-nt overhang at the 3’ end of miRNAs. The N-terminal domain involved in the separation of miRNA-miRNA duplex and the slicer activity of the mRNA (Song, Smith et al.2004). There has been an expansion and duplications of AGO family members during plantevolution (Singh, Gase et al. 2015). The functional diversification of AGOs is indicating sRNAdirected regulatory pathways. The binding preference of AGO and sRNA is mainly assigned by the sequence of sRNA. In Arabidopsis, 10 AGO have been extensively studied (Liu et al. 2014). It has been demonstrated that AtAGO10 like AtAGO1, it recognized distinct structural features in miR165/miR166 duplex than involved by AtGO1. AtAGO10 found to regulate shoot apical meristem by decoying miR165/miR166 and subsequent repression of homeodomain-leucinezipper (HD-ZIP) gene expression (Zhu, Hu et al. 2011). Notably, 22 AGO proteins have been reported in Soybean (Glycine max). It has been found that genome duplication in Soybean resulted such a proliferation of AGOs. For example: its genome encodes two copies of AGO1, AGO2, AGO5, AGO4/9, AGO6 and AGO7 (Xiang Liu 2014). However, the molecular function of the plant AGO genes yet not very clear. There are several miRNA families that are conserved across the vast evolutionary distances from flowering plants to mosses (Cuperus, Fahlgren et al. 2011). It has been observed in another study that miRNA, and its target pairing found to be stable for a prolonged periods of plant evolution. On the flip side, another group demonstrated that conserved plant miRNAs and their targets are to somehow flexible. For instance; miR159 is a highly conserved miRNA that targets not only a subset of MYB mRNAs but also observed to target a non MYB mRNA, SGN-U567133 (Buxdorf, Hendelman et al. 2010). A mutant tomato transgenic line (miR159-resistant line) showed higher level of the SGN-U567133 transcript and exhibited defects in leaf and flower development. This result suggests that miR159 involves in a post-transcriptional regulation. Additionally, it is found to be crucial for the normal tomato development. Recently, the identification of miRNAs in the regulation of photoperiodic pathways in soybean have been reported through high throughput sequencing and qRT-PCR. Six libraries were constructed using Illumina Solexa, for instance; 0, 8, and 16 h under short day treatment, similar time points considered for the long the long day treatment. A total of 163 miRNAs families were reported which covered 318 plant miRNAs, and unclassified 81 novel predicted miRNAs. As expected, significant differences in abundance between short day and long day treatment was observed (Wenbin Li 2015). These findings provided evidence of miRNA in the regulation of flowering time that ultimately affects the seed yield and quality of soybean. The complex regulatory network of miRNA-mRNA interactions during viral infection has been revealed via small RNA seq (sRNA), degradome seq, and genome-wide transcriptome analysis. There has been a total of 253 soybean miRNAs found to be two-folds abundance compared with mock-inoculated control demonstrated through sRNA seq analysis. Among them 105 miRNAs were identified as potential targets of 125 transcripts that has been validated by degradome seq analyses. In addition, 2679 genes were detected via genome wide transcriptomic analysis. These genes have been differentially expressed during infection of soybean mosaic virus and among them 71 genes projected to induce in defense response (Hui Chen 2016). These findings suggested the regulatory role miRNA that governed the target gene expression during viral infection. Furthermore, the regulatory role of microRNAs (miRNAs) during Soybean- Bradyrhizobium japonicum mutualistic association was studied first by Subramanian et al. 2008. They sequenced approximately 350000 small RNAs of soybean root sample which were inoculated with B. japonicum. It helps to detect 20 conserved miRNAs loci based on the similarity to miRNAs in another plant species. In addition, 35 novel miRNAs were identified based on potential hairpin forming precursors in Soybean EST as well as shotgun genomic sequences (Subramanian, Fu et al. 2008). These findings advocated the potential role of miRNAs in the regulation of legumerhizobiumsymbiosis. In another study, 120 hairpin-forming precursor genes have been identified in soybean by Turner et al. In addition, they reported three novel miRNAs for instance; miR160, miR164 and miR393 found to be involved in auxin signaling (Turner, Yu et al. 2012). Moreover, the plant hormone auxin is thought to have a pivotal role in nodule organogenesis in determinate and indeterminate type of nodule. It indicates a redundancy and diversity of miRNAs family members that governs the formation of root nodule. It has been illustrated that auxin receptor gene family hushed by over expressed microRNA393. These plant roots found to be hypersensitive to auxin and yielded normal nodule. This observation advocated that only minimal/reduced auxin signaling is required for determinate nodule development. Likewise, overexpressed microRNA160 hushed a set of repressor auxin response transcription factor. These plant roots were hypersensitive to auxin and observed not to be reluctant in epidermal responses to rhizobia. Notably, it yielded to lower sized nodule primordium (Marie Turner 2013). This observation indicated that auxin hypersensitivity inhibits nodule organogenesis Organ specific expression of profile of miRNA and the potential targets were also studied. Two genes (Glyma10g10240 and Glyma17g05920) which were the target of miR169 but detected to be highly expressed in soybean nodule. Likewise, three potential targets of gma-new-miR13587 demonstrated to be highly expressed in the nodules than in the roots. As expected, gma-newmiR13587 found to be poorly expressed in the nodules than in the roots (Turner, Yu et al. 2012). There was an inverse expression pattern observed in between roots and nodules. Li et al., studied the transgene expression of three novel miRNAs namely, miR482, miR1512, and miR1515 in Soybean. They noticed a significant increase of nodule numbers while root length and later root density were normal in all tested miRNA lines. As expected, there were differential expression of these miRNAs in supernodulating and nonnodulating soybean mutants. They reported that 6 novel miRNAs decoyed 22 predicted target genes. And it was estimated via real time polymerase chain reaction and qRT-PCR (Li, Deng et al. 2010). It advocates that miRNAs have the signatory roles in soybean nodule development. Sequencing of small RNAs and Parallel analysis of RNA ends (PARE) libraries revealed to identify 284 nodule miRNAs, more than 500 target genes, and including 178 novel soybean miRNAs. It has been reported that ENOD93 only found to be expressed in nodule tissue not in other plant parts of Soybean. Ectopic expression of miR393j-3p and RNAi silencing approach to ENOD93 expression showed a significant reduction in nodule formation (Zhe Yan 2015). Therefore, this study showed a list of miRNAs and their potential target of nodulation genes. In the model legume (Medicago truncatula), 25 conserved miRNA families and 100 novel miRNA reads were detected by high-throughput sequencing. The expression of MtHAP2-1 (encodes a CCAAT binding transcription factor) to meristematic zones was restricted by miR169a which is found to be critical for the development of indeterminate type of nodule (Combier, Frugier et al. 2006). In another study, HDZIPIII transcripts were inhibited by overexpression of miR166, it dropped the number of symbiotic nodule and lateral root (Boualem, Laporte et al. 2008). To get insights into key genes of nodule zones, transcript profiles of specific cells/tissues were investigated at different time points from indeterminate nodules of M. truncatulausing laser capture micro dissection. It has been demonstrated from the comprehensive gene expression map that selected genes enriched in different cell/tissue types (Limpens, Moling et al. 2013). These findings indicated that organ specific gene expression could be controlled by the presence or absence of miRNAs. Recently, Agrobacterium rhizogenesmediated hairy root transformation has been applied as tool for exploring cell type specific gene expression in tomato. Cell type or tissue specific promoter introduced into INTACT and TRAP constructs via gateway cloning technology to develop binary vectors. INTACT method used to capture biotin tagged nuceli from specific cell types and TRAP method used for profiling of mRNAs or foot printing of individual ribosomes (Ron 2014). TRAP methodology is not required tissue fixation or single cell suspension. It has been successfully used to date in organisms ranging from D. melanogaster to mice and human cultured cells. Multiple ribosomes or Polyribosomes (polysomes) are engaged in translation on a single mRNA. To evaluate the translation state of an mRNA, ribosomal subunits, ribosomes, and polysomes can be isolated from detergent-treated cell extracts (Heiman, Kulicke et al. 2014). In this study, we would perform polysome isolation deploying gene cassettes ENOD40p:HF-GFP-RPL18 for primordial tissues, and ENOD2p:HF-GFP-RPL18 for parenchymal tissues in Glycine max root nodules that express an epitope tagged version of ribosomal protein L18. Over the last one decade, there has been several microarrays-based studies which characterized transcriptional variations deployed in nodule formation. It has been embedded with couple of shortcomings, for instance; relative late time points study, incomplete representation of plant genes,discrimination of close paralogs, and reduced sensitivity. Lately, next generation sequencingtechnology have widened the horizon of transcription analyses in different legume species to detectsymbiosis induced changes in late nodule developmental stages. Against this backdrop, we areaccentuated to reveal early transcriptional changes induced in determinate type of soybean noduleby Bradyrhizobium japonicum. In determinate type of nodule, two major nodule development zones are formed for instance, the nodule primordium (Npr) in the middle and it is encircled by nodule parenchyma (Npa). At later time point, the Npr converted to N-fixation zone and the Npa contained vascular bundles. Of these facts, it is not clear what early signaling pathways driving the conspicuous development of thenodule zones. In this context, mechanisms regulate the distinct gene expression profiles in Npr andNpa cell types has not understood clearly. The proposed research study is aimed at filling this knowledge gap byillustrating the molecular signatures that paves the way to cellular differentiation in root noduledevelopment in soybean considering four different time points (5 dai, 7 dai, 10 dai& 14 dai). The hypothesisis microRNAs(miRNAs) play important regulatory roles in spatio-temporal expression of their target genesduring nodule developmental in soybean. For example, a gradient of microRNA localizationbetween nodule primordium and parenchyma cells could result in distinct differentiation of thesecell types. To test this hypothesis, one has to obtain both cell type-specific miRNA andtranscriptome (miRNA target) profiles. Since, the majority of miRNA regulation occurs in thecytoplasm, we reasoned that comparison of nuclear and ribosomal transcriptome profiles wouldreveal genes whose expression is potentially regulated by post transcriptional mechanisms such asmiRNA cleavage. Combining this information with cell type-specific miRNA profiles, andto test the above hypothesis and identify key miRNA-target pairs important for nodule celldifferentiation. The use of translating ribosome affinity purification (TRAP) of nodule zonecells, namely from parenchyma and primordial tissues, to obtain cytoplasmic transcriptomes data. Techniques to determine cell type specific expression profiles: TRAP methods TRAP is termed translating ribosome affinity purification, combines cell-type-specific transgene expression with affinity purification of translating ribosomes. It supersedes the need for tissue fixation, and facilitates to study the cell type-specific mRNA profiles of any genetically defined cell type. It has been successfully used to date in organisms ranging from D. melanogaster to mice, and human cultured cells. Multiple ribosomes or Polyribosomes (polysomes) are engaged in translation on a single mRNA. To evaluate the translation state of an mRNA, ribosomal subunits, ribosomes, and polysomes can be isolated from detergent-treated cell extracts. In this study, the polysome isolation using gene cassettes ENOD40p:HF-GFP-RPL18 for primordial tissues, and ENOD2p:HF-GFP-RPL18 for parenchymal tissues in Glycine max root nodules that express an epitope tagged version of ribosomal protein L18 RPL18(Heiman, Kulicke et al. 2014, Ron 2014). Relative abundance and differentially expressed mRNAs profile in two different tissue specific zones would help to understand the effect of regulatory role of miRNAs in cell differentiation and nodule development. References: Axtell, M. J. (2013). "Classification and comparison of small RNAs from plants." Annu Rev PlantBiol 64: 137-159. 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Spontaneous mutations associated with the tomato transcription factors COLORLESS NON-RIPENING (SPL-CNR), NON-RIPENING (NAC-NOR), and RIPENING-INHIBITOR (MADS-RIN) result in fruit that do not undergo the normal hallmarks of ripening but are phenotypically distinguishable. Here, we expanded knowledge of the physiological, molecular, and genetic impacts of the ripening mutations on fruit development beyond ripening. We demonstrated through phenotypic and transcriptome analyses that Cnr fruit exhibit a broad range of developmental defects before the onset of fruit ripening, but fruit still undergo some ripening changes similar to wild type. Thus, Cnr should be considered as a fruit developmental mutant and not just a ripening mutant. Additionally, we showed that some ripening processes occur during senescence in the nor and rin mutant fruit, indicating that while some ripening processes are inhibited in these mutants, others are merely delayed. Through gene expression analysis and direct measurement of hormones, we found that Cnr, nor, and rin have alterations in the metabolism and signaling of plant hormones. Cnr mutants produce more than basal levels of ethylene, while nor and rin accumulate high concentrations of abscisic acid. To determine genetic interactions between the mutations, we created for the first time homozygous double mutants. Phenotypic analyses of the double ripening mutants revealed that Cnr has a strong influence on fruit traits and that combining nor and rin leads to an intermediate ripening mutant phenotype. However, we found that the genetic interactions between the mutations are more complex than anticipated, as the Cnr/nor double mutant fruit has a Cnr phenotype but displayed inhibition of ripening-related gene expression just like nor fruit. Our reevaluation of the Cnr, nor, and rin mutants provides new insights into the utilization of the mutants for studying fruit development and their implications in breeding for tomato fruit quality.

Abstract Although the role of ethylene in tomato (Solanum lycopersicum) fruit ripening has been intensively studied, its role in tomato fruit growth remains poorly understood. In addition, the relationship between ethylene and the developmental factors NON-RIPENING (NOR) and RIPENING INHIBITOR (RIN) during ripening is under debate. Here, we carried out comprehensive genetic analyses of genome-edited mutants of tomato ETHYLENE INSENSITIVE 2 (SlEIN2), four EIN3-like genes (SlEIL1–4), and three EIN3 BINDING F-box protein genes (SlEBF1–3). Both slein2-1 and the high-order sleil mutant (sleil1 sleil2 sleil3/SlEIL3 sleil4) showed reduced fruit size, mainly due to decreased auxin biosynthesis. During fruit maturation, slein2 mutants displayed the complete cessation of ripening, which was partially rescued by slebf1 but not slebf2 or slebf3. We also discovered that ethylene directly activates the expression of the developmental genes NOR, RIN, and FRUITFULL1 (FUL1) via SlEIL proteins. Indeed, overexpressing these genes partially rescued the ripening defects of slein2-1. Finally, the signal intensity of the ethylene burst during fruit maturation was intimately connected with the progression of full ripeness. Collectively, our work uncovers a critical role of ethylene in fruit growth and supports a molecular framework of ripening control in which the developmental factors NOR, RIN, and FUL1 act downstream of ethylene signaling.

Softening is a hallmark of ripening in fleshy fruits, and has both desirable and undesirable implications for texture and postharvest stability. Accordingly, the timing and extent of pre-harvest ripening and associated textural changes following harvest are key targets for improving fruit quality through breeding. Previously, we identified a large effect locus associated with harvest date and firmness in apple (Malus domestica) using genome-wide association studies (GWAS). Here, we present additional evidence that polymorphisms in or around a transcription factor gene, NAC18.1, may cause variation in these traits. First, we confirmed our previous findings with new phenotype and genotype data from ∼800 apple accessions. In this population, we compared a genetic marker within NAC18.1 to markers targeting three other firmness-related genes currently used by breeders (ACS1, ACO1, and PG1), and found that the NAC18.1 marker was the strongest predictor of both firmness at harvest and firmness after 3 months of cold storage. By sequencing NAC18.1 across 18 accessions, we revealed two predominant haplotypes containing the single nucleotide polymorphism (SNP) previously identified using GWAS, as well as dozens of additional SNPs and indels in both the coding and promoter sequences. NAC18.1 encodes a protein that is orthogolous to the NON-RIPENING (NOR) transcription factor, a regulator of ripening in tomato (Solanum lycopersicum). We introduced both NAC18.1 transgene haplotypes into the tomato nor mutant and showed that both haplotypes complement the nor ripening deficiency. Taken together, these results indicate that polymorphisms in NAC18.1 may underlie substantial variation in apple firmness through modulation of a conserved ripening program.

Abstract Melon (Cucumis melo L.) has emerged as an alternative model to study fruit ripening due to the coexistence of climacteric and non-climacteric varieties. The previous characterization of a major QTL ETHQV8.1 sufficient to trigger climacteric ripening in a non-climacteric background allowed the identification within the QTL interval of a negative regulator of ripening CmCTR1-like (MELO3C024518), and a putative DNA demethylase CmROS1 (MELO3C024516), the orthologue of DML2, a DNA demethylase regulating fruit ripening in tomato. To understand the role of these genes in climacteric ripening, we generated homozygous CRISPR knockout mutants of CmCTR1-like and CmROS1 in a climacteric genetic background. The climacteric behavior was altered in both loss-of-function mutants in two summer seasons with an advanced ethylene production profile compared to the climacteric wild type, suggesting a role of both genes in climacteric ripening in melon. Single cytosine methylome analyses of the CmROS1 knockout mutant revealed DNA methylation changes in the promoter regions of key ripening genes as ACS1, ETR1 and ACO1, and ripening associated-transcription factors as NAC-NOR, RIN and CNR, suggesting the importance of CmROS1-mediated DNA demethylation for triggering fruit ripening in melon.

Abstract In tomato (Solanum lycopersicum) and other plants, the photoreceptor UV-RESISTANCE LOCUS 8 regulates plant UV-B photomorphogenesis by modulating the transcription of many genes, the majority of which depends on the transcription factor ELONGATED HYPOCOTYL 5 (HY5). HY5 transcription is induced and then rapidly attenuated by UV-B. However, neither the transcription factors that activate HY5 transcription nor the mechanism for its attenuation during UV-B signaling is known. Here, we report that the tomato B-BOX (BBX) transcription factors SlBBX20 and SlBBX21 interact with SlHY5 and bind to the SlHY5 promoter to activate its transcription. UV-B-induced SlHY5 expression and SlHY5-controlled UV-B responses are normal in slbbx20 and slbbx21 single mutants, but strongly compromised in the slbbx20 slbbx21 double mutant. Surprisingly, UV-B responses are also compromised in lines overexpressing SlBBX20 or SlBBX21. Both SlHY5 and SlBBX20 bind to G-box1 in the SlHY5 promoter. SlHY5 outcompetes SlBBX20 for binding to the SlHY5 promoter in vitro, and inhibits the association of SlBBX20 with the SlHY5 promoter in vivo. Overexpressing 35S:SlHY5-FLAG in the WT background inhibits UV-B-induced endogenous SlHY5 expression. Together, our results reveal the critical role of the SlBBX20/21-SlHY5 module in activating the expression of SlHY5, the gene product of which inhibits its own gene transcription under UV-B, forming an autoregulatory negative feedback loop that balances SlHY5 transcription in plants.

Abstract The pericarp is the predominant tissue determining the structural characteristics of most fruits. However, the molecular and genetic mechanisms controlling pericarp development remain only partially understood. Previous studies have identified that CLASS-II KNOX genes regulate fruit size, shape, and maturation in Arabidopsis thaliana and Solanum lycopersicum. Here we characterized the roles of the Solanum lycopersicum CLASS-II KNOX (TKN-II) genes in pericarp development via a detailed histological, anatomical, and karyotype analysis of the TKN-II mRNA-knockdown (35S:amiR-TKN-II) fruits. We identify that 35S:amiR-TKN-II pericarps contain more cells around their equatorial perimeter and fewer cell layers than the control. In addition, the cell sizes but not the ploidy levels of these pericarps were dramatically reduced. Further, we demonstrate that fruit shape and pericarp layer number phenotypes of the 35S:amiR-TKN-II fruits can be overridden by the procera mutant, known to induce a constitutive response to the plant hormone gibberellin. However, neither the procera mutation nor exogenous gibberellin application can fully rescue the reduced pericarp width and cell size phenotype of 35S:amiR-TKN-II pericarps. Our findings establish that TKN-II genes regulate tomato fruit anatomy, acting via gibberellin to control fruit shape but utilizing a gibberellin-independent pathway to control the size of pericarp cells.

ABSTRACTGenomic data predict that, in addition to oxygen, the bacterial plant pathogenRalstonia solanacearumcan use nitrate (NO3−), nitrite (NO2−), nitric oxide (NO), and nitrous oxide (N2O) as terminal electron acceptors (TEAs). Genes encoding inorganic nitrogen reduction were highly expressed during tomato bacterial wilt disease, when the pathogen grows in xylem vessels. Direct measurements found that tomato xylem fluid was low in oxygen, especially in plants infected by R. solanacearum. Xylem fluid contained ~25 mM NO3−, corresponding to R. solanacearum's optimal NO3−concentration for anaerobic growthin vitro. We tested the hypothesis that R. solanacearum uses inorganic nitrogen species to respire and grow during pathogenesis by making deletion mutants that each lacked a step in nitrate respiration (ΔnarG), denitrification (ΔaniA, ΔnorB, and ΔnosZ), or NO detoxification (ΔhmpX). TheΔnarG,ΔaniA, andΔnorBmutants grew poorly on NO3−compared to the wild type, and they had reduced adenylate energy charge levels under anaerobiosis. While NarG-dependent NO3−respiration directly enhanced growth, AniA-dependent NO2−reduction did not. NO2−and NO inhibited growth in culture, and their removal depended on denitrification and NO detoxification. Thus, NO3−acts as a TEA, but the resulting NO2−and NO likely do not. None of the mutants grew as well as the wild typein planta, and strains lacking AniA (NO2−reductase) or HmpX (NO detoxification) had reduced virulence on tomato. Thus, R. solanacearum exploits host NO3−to respire, grow, and cause disease. Degradation of NO2−and NO is also important for successful infection and depends on denitrification and NO detoxification systems.IMPORTANCEThe plant-pathogenic bacteriumRalstonia solanacearumcauses bacterial wilt, one of the world's most destructive crop diseases. This pathogen's explosive growth in plant vascular xylem is poorly understood. We used biochemical and genetic approaches to show that R. solanacearum rapidly depletes oxygen in host xylem but can then respire using host nitrate as a terminal electron acceptor. The microbe uses its denitrification pathway to detoxify the reactive nitrogen species nitrite (a product of nitrate respiration) and nitric oxide (a plant defense signal). Detoxification may play synergistic roles in bacterial wilt virulence by converting the host's chemical weapon into an energy source. Mutant bacterial strains lacking elements of the denitrification pathway could not grow as well as the wild type in tomato plants, and some mutants were also reduced in virulence. Our results show how a pathogen's metabolic activity can alter the host environment in ways that increase pathogen success.

Abstract Grapevine (Vitis vinifera L.) is one of the most widely cultivated fruit crops because the winemaking industry has huge economic relevance worldwide. Uncovering the molecular mechanisms controlling the developmental progression of plant organs will prove essential for maintaining high-quality grapes, expressly in the context of climate change, which impairs the ripening process. Through a deep inspection of transcriptomic data, we identified VviNAC60, a member of the NAC transcription factor family, as a putative regulator of grapevine organ maturation. We explored VviNAC60 binding landscapes through DNA affinity purification followed by sequencing and compared bound genes with transcriptomics datasets from grapevine plants stably and transiently overexpressing VviNAC60 to define a set of high-confidence targets. Among these, we identified key molecular markers associated with organ senescence and fruit ripening. Physiological, metabolic, and promoter activation analyses showed that VviNAC60 induces chlorophyll degradation and anthocyanin accumulation through the up-regulation of STAY-GREEN PROTEIN 1 (VviSGR1) and VviMYBA1, respectively, with the latter being up-regulated through a VviNAC60-VviNAC03 regulatory complex. Despite sharing a closer phylogenetic relationship with senescence-related homologues to the NAC transcription factor AtNAP, VviNAC60 complemented the non-ripening(nor) mutant phenotype in tomato (Solanum lycopersicum), suggesting a dual role as an orchestrator of both ripening- and senescence-related processes. Our data support VviNAC60 as a regulator of processes initiated in the grapevine vegetative- to mature-phase organ transition and therefore as a potential target for enhancing the environmental resilience of grapevine by fine-tuning the duration of the vegetative phase.

Abstract Plant and animal intracellular nucleotide-binding and leucine-rich repeat (NLR) receptors play important roles in sensing pathogens and activating defense signaling. However, the molecular mechanisms underlying the activation of host defense signaling by NLR proteins remain largely unknown. Many studies have determined that the coil–coil (CC) or Toll and interleukin-1 receptor/resistance protein (TIR) domain of NLR proteins and their dimerization/oligomerization are critical for activating downstream defense signaling. In this study, we demonstrated that, in tomato, the nucleotide-binding (NB) domain Sw-5b NLR alone can activate downstream defense signaling, leading to elicitor-independent cell death. Sw-5b NB domains can self-associate, and this self-association is crucial for activating cell death signaling. The self-association was strongly compromised after the introduction of a K568R mutation into the P-loop of the NB domain. Consequently, the NBK568R mutant induced cell death very weakly. The NBCΔ20 mutant lacking the C-terminal 20 amino acids can self-associate but cannot activate cell death signaling. The NBCΔ20 mutant also interfered with wild-type NB domain self-association, leading to compromised cell death induction. By contrast, the NBK568R mutant did not interfere with wild-type NB domain self-association and its ability to induce cell death. Structural modeling of Sw-5b suggests that NB domains associate with one another and likely participate in oligomerization. As Sw-5b-triggered cell death is dependent on helper NLR proteins, we propose that the Sw-5b NB domain acts as a nucleation point for the assembly of an oligomeric resistosome, probably by recruiting downstream helper partners, to trigger defense signaling.

AbstractPlants rely on innate immunity to perceive and ward off microbes and pests, and are able to overcome the majority of invading microorganisms. Even so, specialized pathogens overcome plant defenses, posing a persistent threat to crop and food security worldwide, raising the need for agricultural products with broad, efficient resistance. Here we report a specific mutation in a tomato (S. lycopersicum) helper nucleotide-binding domain leucine-rich repeat H-NLR, SlNRC4a, which results in gain of function constitutive basal defense activation, in absence of PRR activation. Knockout of the entire NRC4 clade in tomato was reported to compromise Rpi-blb2 mediated immunity. The SlNRC4a mutant reported here possesses enhanced immunity and disease resistance to a broad-spectrum of pathogenic fungi, bacteria and pests, while lacking auto-activated HR or negative effects on plant growth and crop yield, providing promising prospects for agricultural adaptation in the war against plant pathogens that decrease productivity.