Academic literature on the topic 'Rha4 locus'

ABSTRACT Rhodosporidium toruloides is a heterothallic, bipolar, red yeast that belongs to the Sporidiobolales, an order within a major lineage of basidiomycetes, the Pucciniomycotina. In contrast to other basidiomycetes, considerably less is known about the nature of the mating type (MAT) loci that control sexual reproduction in this lineage. Three genes (RHA1, RHA2, and RHA3) encoding precursors of the MAT A1 pheromone (rhodotorucine A) were previously identified and formed the basis for a genome walking approach that led to the identification of additional MAT genes in complementary mating strains of R. toruloides. Two mating type-specific alleles encoding a p21-activated kinase (PAK; Ste20 homolog) were found between the RHA2 and RHA3 genes, and identification in MAT A2 strains of a gene encoding a presumptive pheromone precursor enabled prediction of the structure of rhodotorucine a. In addition, a putative pheromone receptor gene (STE3 homolog) was identified upstream of RHA1. Analyses of genomic data from two closely related species, Sporobolomyces roseus and Sporidiobolus salmonicolor, identified syntenic regions that contain homologs of all the above-mentioned genes. Notably, six novel pheromone precursor genes were uncovered, which encoded, similarly to the RHA genes, multiple tandem copies of the peptide moiety. This suggests that this structure, which is unique among fungal lipopeptide pheromones, seems to be prevalent in red yeasts. Species comparisons provided evidence for a large, multigenic MAT locus structure in the Sporidiobolales, but no putative homeodomain transcription factor genes (which are present in all basidiomycetous MAT loci characterized thus far) could be found in any of the three species in the vicinity of the MAT genes identified.

ABSTRACTRhamnose catabolism inRhizobium leguminosarumwas found to be necessary for the ability of the organism to compete for nodule occupancy. Characterization of the locus necessary for the catabolism of rhamnose showed that the transport of rhamnose was dependent upon a carbohydrate uptake transporter 2 (CUT2) ABC transporter encoded byrhaSTPQand on the presence of RhaK, a protein known to have sugar kinase activity. A linker-scanning mutagenesis analysis ofrhaKshowed that the kinase and transport activities of RhaK could be separated genetically. More specifically, two pentapeptide insertions defined by the allelesrhaK72andrhaK73were able to uncouple the transport and kinase activities of RhaK, such that the kinase activity was retained, but cells carrying these alleles did not have measurable rhamnose transport rates. These linker-scanning alleles were localized to the C terminus and N terminus of RhaK, respectively. Taken together, the data led to the hypothesis that RhaK might interact either directly or indirectly with the ABC transporter defined byrhaSTPQ. In this work, we show that both N- and C-terminal fragments of RhaK are capable of interacting with the N-terminal fragment of the ABC protein RhaT using a 2-hybrid system. Moreover, if RhaK fragments carrying either therhaK72orrhaK73allele were used, this interaction was abolished. Phylogenetic and bioinformatic analysis of the RhaK fragments suggested that a conserved region in the N terminus of RhaK may represent a putative binding domain. Alanine-scanning mutagenesis of this region followed by 2-hybrid analysis revealed that a substitution of any of the conserved residues greatly affected the interaction between RhaT and RhaK fragments, suggesting that the sugar kinase RhaK and the ABC protein RhaT interact directly.IMPORTANCEABC transporters involved in the transport of carbohydrates help define the overall physiological fitness of bacteria. The two largest groups of transporters are thecarbohydrateuptaketransporter classes 1 and 2 (CUT1 and CUT2, respectively). This work provides the first evidence that a kinase that is necessary for the catabolism of a sugar can directly interact with a domain from the ABC protein that is necessary for its transport.

Abstract During a humoral immune response, B cells produce alternative immunoglobulin isotypes (IgG, IgA, IgE) through class switch recombination (CSR). Initiation of CSR requires AID-mediated deamination of switch (S) regions in the immunoglobulin heavy chain (IgH) locus. Although G-quadruplex(G4)-forming S region RNAs bind AID and localize AID to S region DNA sequences, the molecular mechanism that regulates the transition of AID binding from RNA to DNA remains uncharacterized. Highly stable G4 structures necessitate helicases to unwind the G-G hydrogen bonds, which in turn may permit the base-pairing of the S transcript to the complementary DNA sequence in the IgH locus during CSR. An S region RNA pull-down assay identified the RNA Helicase associated with AU-rich element (RHAU), a DEAH-box RNA helicase, as an S transcript binding protein. To examine the role of RHAU in CSR, we genetically deleted a floxed RHAU allele in B cells of mice using CD23-Cre. Conditional deletion of RHAU in B cells reduced CSR in vivo and in vitro to approximately 50% of wild-type controls. This data suggests that RHAU plays an important role in CSR. We hypothesize that RHAU unwinds G4 in S transcripts to facilitate the handoff of AID from G4-RNA to S region DNA during CSR. Supported by The National Cancer Institute (2U54CA132378) and The National Institute of General Medical Sciences (1SC1GM132035-01)

ABSTRACT Strains of Rhizobium leguminosarum which are unable to catabolize l-rhamnose, a methyl-pentose sugar, are compromised in the ability to compete for nodule occupancy versus wild-type strains. Previous characterization of the 11-kb region necessary for the utilization of rhamnose identified a locus carrying catabolic genes and genes encoding the components of an ABC transporter. Genetic evidence suggested that the putative kinase RhaK carried out the first step in the catabolism of rhamnose. Characterization of this kinase led to the observation that strains carrying rhamnose kinase mutations were unable to transport rhamnose into the cell. The absence of a functional rhamnose kinase did not stop the transcription and translation of the ABC transporter components. By developing an in vitro assay for RhaK activity, we have been able to show that (i) RhaK activity is consistent with RhaK phosphorylating rhamnose and (ii) biochemical activity of RhaK is necessary for rhamnose transport.

Summary Soybean cyst nematode (SCN, Heterodera glycines) is a devastating plant-parasitic nematode worldwide. Two SCN races, race 4 (HG Type 1.2.3.5.6.7) and race 5 (HG Type 2.5.7), with increased virulence were previously identified in Northeast China. To obtain new resistance sources to these SCN populations, the response of 62 genotypes, including 51 local varieties and breeding lines, and 11 indicator lines for SCN race and HG Type identification, were evaluated. Four new primers in the regions of two loci of GmSHMT08 (Rhg4) and GmSNAP18 (rhg1) were designed for PCR amplification and subsequent sequencing to characterise haplotypes instead of genome resequencing. Results indicated three haplotypes among 51 local genotypes; there were 26 lines in Haplotype I carrying both the rhg1-a and Rhg4-a resistant loci as in ‘Peking’, 13 lines in Haplotype II containing only the resistant rhg1-a locus but Rhg4-b susceptible loci, and 12 lines in Haplotype III with rhg1-c and Rhg4-b susceptible loci. Interestingly, there was no ‘PI 88788’-type resistance identified in Northeast China, although it accounts for 90% of sources in the USA. Two local breeding lines in Haplotype I displayed resistance to both SCN races. The resistance lines carried higher copy number (>1) of the tandem duplication at the rhg1 locus compared with susceptible lines (⩽1). The combination of the two microsatellite markers, Sat_162 on Chr 8 and 590 on Chr 18, distinguished the three haplotypes and predicted the resistance/susceptibility for SCN race 5. The knowledge of the phenotypes and molecular characteristics of 51 local breeding lines in Northeast China will accelerate the utilisation of sources for broad-based SCN resistance and marker-assisted selection.

Clostridium difficile is a bacterial pathogen that causes major health challenges worldwide. It has a well-characterized surface (S)-layer, a para-crystalline proteinaceous layer surrounding the cell wall. In many bacterial and archaeal species, the S-layer is glycosylated, but no such modifications have been demonstrated in C. difficile. Here, we show that a C. difficile strain of S-layer cassette type 11, Ox247, has a complex glycan attached via an O-linkage to Thr-38 of the S-layer low-molecular-weight subunit. Using MS and NMR, we fully characterized this glycan. We present evidence that it is composed of three domains: (i) a core peptide–linked tetrasaccharide with the sequence -4-α-Rha-3-α-Rha-3-α-Rha-3-β-Gal-peptide; (ii) a repeating pentasaccharide with the sequence -4-β-Rha-4-α-Glc-3-β-Rha-4-(α-Rib-3-)β-Rha-; and (iii) a nonreducing end–terminal 2,3 cyclophosphoryl-rhamnose attached to a ribose-branched sub-terminal rhamnose residue. The Ox247 genome contains a 24-kb locus containing genes for synthesis and protein attachment of this glycan. Mutations in genes within this locus altered or completely abrogated formation of this glycan, and their phenotypes suggested that this S-layer modification may affect sporulation, cell length, and biofilm formation of C. difficile. In summary, our findings indicate that the S-layer protein of SLCT-11 strains displays a complex glycan and suggest that this glycan is required for C. difficile sporulation and control of cell shape, a discovery with implications for the development of antimicrobials targeting the S-layer.

ABSTRACT Rhizobium leguminosarum bv. trifolii mutants unable to catabolize the methyl-pentose rhamnose are unable to compete effectively for nodule occupancy. In this work we show that the locus responsible for the transport and catabolism of rhamnose spans 10,959 bp. Mutations in this region were generated by transposon mutagenesis, and representative mutants were characterized. The locus contains genes coding for an ABC-type transporter, a putative dehydrogenase, a probable isomerase, and a sugar kinase necessary for the transport and subsequent catabolism of rhamnose. The regulation of these genes, which are inducible by rhamnose, is carried out in part by a DeoR-type negative regulator (RhaR) that is encoded within the same transcript as the ABC-type transporter but is separated from the structural genes encoding the transporter by a terminator-like sequence. RNA dot blot analysis demonstrated that this terminator-like sequence is correlated with transcript attenuation only under noninducing conditions. Transport assays utilizing tritiated rhamnose demonstrated that uptake of rhamnose was inducible and dependent upon the presence of the ABC transporter at this locus. Phenotypic analyses of representative mutants from this locus provide genetic evidence that the catabolism of rhamnose differs from previously described methyl-pentose catabolic pathways.

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is the foremost pest of soybean (Glycine max L. Merr.). The rhg1 allele on linkage group (LG) G and the Rhg4 allele on LG A2 are important in conditioning resistance. Markers closely linked to the Rhg4 locus were used previously to screen a library of bacterial artificial chromosome (BAC) clones from susceptible 'Williams 82' and identified a single 150-kb BAC, Gm_ISb001_056_G02 (56G2). End-sequenced subclones positioned onto a restriction map provided landmarks for identifying the corresponding region from a BAC library from accession PI 437654 with broad resistance to SCN. Seventy-three PI 437654 BACs were assigned to contigs based upon HindIII restriction fragment profiles. Four contigs represented the PI 437654 counterpart of the 'Williams 82' BAC, with PCR assays connecting these contigs. Some of the markers on the PI 437654 contigs are separated by a greater physical distance than in the 'Williams 82' BAC and some primers amplify bands from BACs in the mid-portion of the connected PI 437654 BAC contigs that are not amplified from the 'Williams 82' BAC. These observations suggest that there is an insertion in the PI 437654 genome relative to the 'Williams 82' genome in the Rhg4 region.Key words: BAC, deletion, insertion, resistance gene, soybean cyst nematode.

Barley (Hordeum vulgare) is a widely grown, valuable cereal crop that is affected by various pests including nematodes. The cereal cyst nematode (CCN) Heterodera avenae is the most widely distributed and damaging species of nematodes to cereal crops grown in temperate regions, including Australia, and is estimated to cause global annual losses of around $160 billion. The use of cultivars resistant to H. avenae is the preferred approach for nematode management and four resistance loci, Rha1, Rha2, Rha3 and Rha4, have been mapped. The Rha4 locus was mapped in the Galleon/Haruna Nijo population to chromosome 5H and since Rha2 and Rha4 provide the most effective resistance against the Australian H. avenae pathotype Ha13 they have been widely used in barley breeding. Despite CCN resistance loci having been mapped in barley and other cereals, no resistance genes have been isolated and characterized. Recently both Rha2 and Rha4 have been fine-mapped and near-diagnostic markers have been developed to provide simple tools for selection. Fine-mapping indicates that approximately 105 genes are linked to the Rha4 locus, including cell wall-related genes predicted to encode (1-4)-β-xylan endohydrolases, also known as xylanases. This thesis reports on experiments that were undertaken to better understand the resistance conferred by Rha4 and to investigate the functions of the xylanases as prime candidate genes. The xylanase genes at the Rha4 locus were cloned and analysed for allelic differences between sequences from the cultivars Sloop (susceptible) and Flagship (Rha4-resistant). Although genes X1 and X2 have been characterised, the X3 gene has not been well described previously. The genomic sequences were used in reciprocal transformation experiments where, under the control of the 35S promoter, the genes from Sloop were transformed into a Flagship background, and vice versa. Tube tests were used to investigate any changes in nematode infection responses, and therefore resistance status, but no significant alterations were detected. However, overexpression of the xylanase genes proved to be detrimental to the overall health of the plant. The xylanases were concomitantly heterologously expressed in Escherichia coli and the X2 protein was characterized in terms of substrate preference and catalytic rate. In more general approaches not directly linked to Rha4 genotype, the natural variation in root polysaccharide amount and distribution, with a focus on (1,3;1,4)-β-glucan and arabinoxylan, was surveyed in a selection of susceptible and resistant barley cultivars. The effect of changes in amount of (1,3;1,4)-β-glucan was also examined through infection of the betaglucanless mutant and transgenic lines carrying either the overexpressed or silenced (1,3;1,4)-β-glucan synthase CslF6 gene. Variable amounts of (1,3;1,4)-β-glucan did not correlate to rates of nematode infection and no clear patterns of polysaccharide profiles could be linked to susceptible or resistant cultivar status. Finally, RNA sequencing was used to profile transcript changes in nematode infected and control Sloop and Flagship roots up to 24 days post inoculation. The patterns of the 105 genes linked to the Rha4 locus were extracted and a set of 27 genes which showed significant fold changes across the time course were profiled. Of these, three strong candidate genes were selected which are differentially expressed in the two cultivars and are involved in biochemical pathways that are feasibly linked to resistance mechanisms. Their putative roles in conferring resistance and how this might be tested were discussed.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food & Wine, 2019

Cereal cyst nematodes (CCN, Heterodera avenae) are obligate soil-borne pathogens of cereal crops such as barley (Hordeum vulgare) that can reduce plant vigour and crop yield. Juvenile nematodes infect roots, establish feeding sites in root vascular tissue and become sedentary. Female nematodes mature into egg-filled cysts, which remain in the soil. Production of susceptible cultivars allows the numbers of cysts to increase, increasing the risk of damage to future crops. Some barley cultivars are resistant. Their roots can be infected, but few nematodes mature and form cysts. The focus of this thesis is the resistance conferred by the Rha2 locus on chromosome 2H of barley. By undertaking genotyping-by-sequencing of the resistant cultivars Sloop SA and Sloop VIC, their susceptible progenitor Sloop and other material, new DNA sequence polymorphisms were discovered on chromosome 2H. Marker assays were designed for these polymorphisms and assayed on two populations (Clipper/Sahara 3771 and Chebec/Harrrington) in which Rha2 had been previously mapped. An initial candidate region of 5,077 kbp was defined on the barley genome assembly. In order to obtain new recombinants and develop near-isogenic lines, Sloop SA was backcrossed to Sloop. Over 9,000 BC2F2 seeds were genotyped. A detailed genetic map of the candidate region was made by genotyping and phenotyping 64 selected BC2F3 families. The candidate region was narrowed to 978 kbp. That region of the genome assembly has 19 predicted genes. Markers in the region were evaluated on a range of barley germplasm and two markers were found to be diagnostic of CCN resistance. An RNA-seq experiment was conducted with root tissue sampled over a period of 28 days. The samples comprise non-inoculated control plants of the susceptible cultivar Sloop and inoculated plants of Sloop and its resistant derivatives Sloop SA and Sloop VIC. Of the 19 predicted genes in the candidate region, one gene (HORVU2Hr1G097780), which is annotated as encoding a tonoplast intrinsic protein, exhibited differential expression between the inoculated resistant cultivars and the susceptible cultivar. Further research is required for the functional characterisation of this gene. Interaction between cereal cyst nematodes and barley roots was also investigated by using laser ablation tomography to scan infected segments. On average, feeding sites in the roots of susceptible plants were smaller than those in the roots of resistant plants. The feeding sites in the roots of susceptible plants were surrounded by multiple dense layers of small cells. In contrast, the feeding sites in the roots of resistant plants were surrounded by layers of larger cells. This work presents a detailed genetic map of the Rha2 region of chromosome 2H, including two markers that appear to be diagnostic of resistance, the results of a transcriptomic experiment to explore differentially regulated genes, including candidate resistance genes. Laser ablation tomography was conducted on infected root tissue. Feeding site structure differed between a susceptible cultivar and its resistant derivative, including a smaller volume for the latter. The outcomes of this thesis research may lead to identification of the causal Rha2 resistance gene against cereal cyst nematode pathotype Ha13.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2019