Gene-for-gene Research Articles

The race structure of Leptosphaeria maculans in western Canada between 2012 and 2014 and its influence on blackleg of canola

Field surveys indicate blackleg of canola has increased in western Canada since 2010. Earlier studies showed changes in the pathogen (Leptosphaeria maculans (Sowerby) P.Karst.; Lm) population between 2007 and 2010–2011. In this study, Lm isolates were collected from trap plots (591) and/or commercial fields (372) on the prairies between 2012 and 2014, and tested for the profile of avirulence (Av) genes on a set of host differentials for 10 Lm Av alleles. Up to 35 ‘Westar’ trap plots were set up each year, and commercial fields were surveyed in the same area in 2012 and 2013. A similar trend was observed for Av profile between the two Lm populations; Av1, Av3, Av9 and AvLep2 were at low or very low levels, while Av2, Av4, Av6 and Av7 were present in >60% of the isolates. A total of 82 races were identified in these Lm populations, suggesting a greater diversity than those reported elsewhere. Margalef and Simpson indices also confirmed the greater genetic diversity in Lm. In fact, for each known R gene, there is at least one virulent race already existing in the Lm population. The races carrying Av2, Av4, Av6 or Av7 accounted for >70% of the Lm population, indicating that an R gene corresponding to any of these Av alleles may be effective against blackleg on the prairies. However, Av results alone appeared insufficient for explaining different levels of blackleg observed in canola fields; additional factors, possibly including crop rotation and local weather, may also play a role.

Evolution of views on plant immunity: from Flor’s “gene-for-gene” theory to the “zig-zag model” developed by Jones and Dangl

The study of plant defence mechanisms in response to pathogens in the mid-20th century resulted in Harold Flor’s gene-for-gene interaction hypothesis, which became recognised as central to the study of phytoimmunity. According to this theory, the outcome of interactions in plant – pathogen phytopathosystems – i.e. compatibility or incompatibility – is controlled genetically in interacting organisms and determined by the presence of specific genes in both pathogen and plant: resistance genes in the plant and avirulence genes in pathogen. The latest achievements in phytoimmunology, obtained with the help of modern molecular biology and bioinformatics methods, have made a significant contribution to the classical understanding of plant immunity and provided grounds for a modern concept of phytoimmunity consisting in the “zig-zag model” developed by Jonathan Jones and Jefferey Dangl. Plant immunity is currently understood as being determined by an innate multi-layer immune system involving various structures and mechanisms of specific and non-specific immunity. Recognition by plant membrane receptors of conservative molecular patterns associated with microorganisms, as well as molecules produced during cell wall disruption by pathogen hydrolytic enzymes forms a basic non-specific immune response in the plant. Detection of pathogen effector molecules by plant intra-cellular receptors triggers a specific effector-triggered immunity, resulting in the development of the hypersensitive response, systemic resistance and immune memory of the plant. Virulence factors and pathogen attack strategies on the one hand, and mechanisms of plant immune protection on the other, are the result of one form of constant co-evolution, often termed an “evolutionary arms race”. This paper discusses the main principles of Flor's classical “gene-for-gene interaction” theory as well as the molecular-genetic processes of plant innate immunity, their mechanisms and participants in light of contemporary achievements in phytoimmunology.

The Brassica napus wall-associated kinase-like (WAKL) gene Rlm9 provides race-specific blackleg resistance.

SUMMARYIn plants, race‐specific defence against microbial pathogens is facilitated by resistance (R) genes which correspond to specific pathogen avirulence genes. This study reports the cloning of a blackleg R gene from Brassica napus (canola), Rlm9, which encodes a wall‐associated kinase‐like (WAKL) protein, a newly discovered class of race‐specific plant RLK resistance genes. Rlm9 provides race‐specific resistance against isolates of Leptosphaeria maculans carrying the corresponding avirulence gene AvrLm5‐9, representing only the second WAKL‐type R gene described to date. The Rlm9 protein is predicted to be cell membrane‐bound and while not conclusive, our work did not indicate direct interaction with AvrLm5‐9. Rlm9 forms part of a distinct evolutionary family of RLK proteins in B. napus, and while little is yet known about WAKL function, the Brassica–Leptosphaeria pathosystem may prove to be a model system by which the mechanism of fungal avirulence protein recognition by WAKL‐type R genes can be determined.

Suppression of wheat blast resistance by an effector of Pyricularia oryzae is counteracted by a host specificity resistance gene in wheat.

Wheat blast caused by the Triticum pathotype of Pyricularia oryzae poses a serious threat to wheat production in South America and Asia and is now becoming a pandemic disease. Here, we show that Rmg8, a promising wheat gene for resistance breeding, is suppressed by PWT4, an effector gene of P.oryzae, and in turn that the suppression is counteracted by Rwt4, a wheat gene recognizing PWT4. When PWT4 was introduced into a wheat blast isolate carrying AVR-Rmg8 (an avirulence gene corresponding to Rmg8), PWT4 suppressed wheat resistance conferred by Rmg8. PWT4 did not alter the expression of AVR-Rmg8, but higher expression of PWT4 led to more efficient suppression. This suppression was observed in rwt4 carriers, but not in Rwt4 carriers, indicating that it is counteracted by Rwt4. PWT4 was assumed to have been horizontally transferred from a weed-associated cryptic species, P.pennisetigena, to an Avena isolate of P.oryzae in Brazil. This implies a potential risk of the acquisition of PWT4 by the wheat blast fungus and the 'breakdown' of Rmg8. We suggest that Rmg8 should be introduced together with Rwt4 into a wheat cultivar when it is used for resistance breeding.

Effectiveness of the Wheat Blast Resistance Gene Rmg8 in Bangladesh Suggested by Distribution of an AVR-Rmg8 Allele in the Pyricularia oryzae Population.

Wheat blast caused by the Triticum pathotype of Pyricularia oryzae was first reported in 1985 in Brazil and recently spread to Bangladesh. We tested whether Rmg8 and RmgGR119, recently identified resistance genes, were effective against Bangladeshi isolates of the pathogen. Common wheat accessions carrying Rmg8 alone (IL191) or both Rmg8 and RmgGR119 (GR119) were inoculated with Brazilian isolates (Br48, Br5, and Br116.5) and Bangladeshi isolates (T-108 and T-109). Br48, T-108, and T-109 carried the eI type of AVR-Rmg8 (the avirulence gene corresponding to Rmg8) while Br5 and Br116.5 carried its variants, eII and eII' types, respectively. Detached primary leaves of IL191 and GR119 were resistant to all isolates at 25°C. At a higher temperature (28°C), their resistance was still effective against the eI carriers but was reduced to a low level against the eII/eII' carriers. A survey of databases and sequence analyses revealed that all Bangladeshi isolates carried the eI type which induced a higher level of resistance than the eII/eII' types. The resistance of IL191 (Rmg8/-) to the eI carriers was maintained even at the heading stage and at the higher temperature. In addition, GR119 (Rmg8/RmgGR119) displayed higher levels of resistance than IL191 at this stage. These results suggest that Rmg8 combined with RmgGR119 will be useful in breeding for resistance against wheat blast in Bangladesh.

Integrated Analysis of Gene Expression, SNP, InDel, and CNV Identifies Candidate Avirulence Genes in Australian Isolates of the Wheat Leaf Rust Pathogen Puccinia triticina.

The leaf rust pathogen, Puccinia triticina (Pt), threatens global wheat production. The deployment of leaf rust (Lr) resistance (R) genes in wheat varieties is often followed by the development of matching virulence in Pt due to presumed changes in avirulence (Avr) genes in Pt. Identifying such Avr genes is a crucial step to understand the mechanisms of wheat-rust interactions. This study is the first to develop and apply an integrated framework of gene expression, single nucleotide polymorphism (SNP), insertion/deletion (InDel), and copy number variation (CNV) analysis in a rust fungus and identify candidate avirulence genes. Using a long-read based de novo genome assembly of an isolate of Pt (‘Pt104’) as the reference, whole-genome resequencing data of 12 Pt pathotypes derived from three lineages Pt104, Pt53, and Pt76 were analyzed. Candidate avirulence genes were identified by correlating virulence profiles with small variants (SNP and InDel) and CNV, and RNA-seq data of an additional three Pt isolates to validate expression of genes encoding secreted proteins (SPs). Out of the annotated 29,043 genes, 2392 genes were selected as SP genes with detectable expression levels. Small variant comparisons between the isolates identified 27–40 candidates and CNV analysis identified 14–31 candidates for each Avr gene, which when combined, yielded the final 40, 64, and 69 candidates for AvrLr1, AvrLr15, and AvrLr24, respectively. Taken together, our results will facilitate future work on experimental validation and cloning of Avr genes. In addition, the integrated framework of data analysis that we have developed and reported provides a more comprehensive approach for Avr gene mining than is currently available.

UhAVR1, an HR-Triggering Avirulence Effector of Ustilago hordei, Is Secreted via the ER-Golgi Pathway, Localizes to the Cytosol of Barley Cells during in Planta-Expression, and Contributes to Virulence Early in Infection.

The basidiomycete Ustilago hordei causes covered smut disease of barley and oats. Virulence effectors promoting infection and supporting pathogen lifestyle have been described for this fungus. Genetically, six avirulence genes are known and one codes for UhAVR1, the only proven avirulence effector identified in smuts to date that triggers complete immunity in barley cultivars carrying resistance gene Ruh1. A prerequisite for resistance breeding is understanding the host targets and molecular function of UhAVR1. Analysis of this effector upon natural infection of barley coleoptiles using teliospores showed that UhAVR1 is expressed during the early stages of fungal infection where it leads to HR triggering in resistant cultivars or performs its virulence function in susceptible cultivars. Fungal secretion of UhAVR1 is directed by its signal peptide and occurs via the BrefeldinA-sensitive ER–Golgi pathway in cell culture away from its host. Transient in planta expression of UhAVR1 in barley and a nonhost, Nicotiana benthamiana, supports a cytosolic localization. Delivery of UhAVR1 via foxtail mosaic virus or Pseudomonas species in both barley and N. benthamiana reveals a role in suppressing components common to both plant systems of Effector- and Pattern-Triggered Immunity, including necrosis triggered by Agrobacterium-delivered cell death inducers.

Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq).

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, significantly hampers potato production. Recently, a new Resistance to Phytophthora infestans (Rpi) gene, Rpi‐amr1, was cloned from a wild Solanum species, Solanum americanum. Identification of the corresponding recognized effector (Avirulence or Avr) genes from P. infestans is key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance. To identify the P. infestans effector recognized by Rpi‐amr1, we screened available RXLR effector libraries and used long read and cDNA pathogen‐enrichment sequencing (PenSeq) on four P. infestans isolates to explore the untested effectors. Using single‐molecule real‐time sequencing (SMRT) and cDNA PenSeq, we identified 47 highly expressed effectors from P. infestans, including PITG_07569, which triggers a highly specific cell death response when transiently coexpressed with Rpi‐amr1 in Nicotiana benthamiana, suggesting that PITG_07569 is Avramr1. Here we demonstrate that long read and cDNA PenSeq enables the identification of full‐length RXLR effector families and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RXLR effector family that is associated with the recognition by Rpi‐amr1.

Development of a specific marker for detection of a functional AvrLm9 allele and validating the interaction between AvrLm7 and AvrLm9 in Leptosphaeria maculans.

Blackleg, which is caused by the fungus Leptosphaeria maculans (L. maculans), is a major disease of canola in western Canada and worldwide. Long-term use of one source of resistance could cause the breakdown of its effectiveness. Therefore, appropriate use of R genes is very important, and knowledge about the distribution of avirulence genes is a prerequisite for effectively deploying resistance. Of the 14 avirulence genes identified in L. maculans, AvrLm5 and AvrLm9 were recognized as the two alleles of the same gene based on two single nucleotide polymorphisms, C85T and G164A/C. In this study, a specific marker was developed to identify AvrLm5 and AvrLm9 based on two single nucleotide polymorphisms, C85T and G164A/C, which are responsible for the function of AvrLm9. The specific marker can be used to discriminate the AvrLm9 from avrLm9 accurately in L. maculans isolates, which is consistent with inoculation tests in isolates without AvrLm4-7. This specific marker was used to screen 1229 isolates collected from fields in the years 2014 through 2016 in Manitoba. From 68 to 84% of the isolates were found to contain the AvrLm9 allele; while 4-7% of them were avirulent on the variety Goéland with Rlm9 loci. Furthermore, no isolates having both AvrLm9 and AvrLm7 were detected using a cotyledon test, while 67% to 84% of isolates contained both avirulence genes via PCR detection, implying suppression of AvrLm9 by AvrLm7. In addition, avirulence gene profiles of the other 10 avirulence alleles were examined with the 1229 isolates using cotyledon tests or PCR amplifications. Taken together, this research enables the fast identification of AvrLm5/9, provides the Avr genes' landscape of western Canada and elaborates the relationship between AvrLm9 and AvrLm7 using isolates from grower fields.

Novel Variation and Evolution of AvrPiz-t of Magnaporthe oryzae in Field Isolates.

The product of the avirulence (Avr) gene of Magnaporthe oryzae can be detected by the product of the corresponding resistance (R) gene of rice and activates immunity to rice mediated by the R gene. The high degree of variability of M. oryzae isolates in pathogenicity makes the control of rice blast difficult. That resistance of the R gene in rice has been lost has been ascribed to the instability of the Avr gene in M. oryzae. Further study on the variation of the Avr genes in M. oryze field isolates may yield valuable information on the durable and effective deployment of R genes in rice production areas. AvrPiz-t and Piz-t are a pair of valuable genes in the Rice-Magnaporthe pathosystem. AvrPiz-t is detectable by Piz-t and determines the effectiveness of Piz-t. To effectively deploy the R gene Piz-t, the distribution, variation, and evolution of the corresponding Avr gene AvrPiz-t were found among 312 M. oryzae isolates collected from Yunnan rice production areas of China. PCR amplification and pathogenicity assays of AvrPiz-t showed that 202 isolates (64.7%) held AvrPiz-t alleles and were avirulent to IRBLzt-T (holding Piz-t). There were 42.3–83.3% avirulent isolates containing AvrPiz-t among seven regions in Yunnan Province. Meanwhile, 11 haplotypes of AvrPiz-t encoding three novel AvrPiz-t variants were identified among 100 isolates. A 198 bps insertion homologous to solo-LTR of the retrotransposon inago2 in the promoter region of AvrPiz-t in one isolate and a frameshift mutation of CDS in another isolate were identified among 100 isolates, and those two isolates had evolved to virulent from avirulent. Synonymous mutation and non-AUG-initiated N-terminal extensions keeps the AvrPiz-t gene avirulence function in M. oryzae field isolates in Yunnan. A haplotype network showed that H3 was an ancestral haplotype. Structure variance for absence (28.2%) or partial fragment loss (71.8%) of AvrPiz-t was found among 39 virulent isolates and may cause the AvrPiz-t avirulence function to be lost. Overall, AvrPiz-t evolved to virulent from avirulent forms via point mutation, retrotransposon, shift mutation, and structure variance under field conditions.

In-silico identification and differential expression of putative disease resistance-related genes within the collinear region of Brassica napus blackleg resistance locus LepR2\u2019 in Brassica oleracea

Blackleg disease, caused by Leptosphaeria maculans, greatly affects the production of cabbage (Brassica oleracea). However, definitive R-gene(s) are yet to be identified in this crop. In contrast, a number of R-loci have been identified in A- or B-genome crops. Identification of few resistant cabbage genotypes indicates the presence of R-genes in this C-genome crop. High ancestral synteny between Brassica genomes suggests that the collinear regions of known A- or B-genome R-loci may also contain functional R-genes in the C-genome. Strong resistance was observed in the cotyledons of cabbage inbred line SCNU-98 against two L. maculans isolates, 03–02 s and 00–100 s. We investigated the collinear region of the Brassica napus blackleg resistance locus LepR2’ in B. oleracea since both isolates of L. maculans contain corresponding avirulence genes. The locus was collinear to a 5.8 Mbp genomic segment of B. oleracea chromosome C09 containing 13 genes that have putative disease resistance-related domains. High expression of genes Bo9g117290 and Bo9g111510 against isolate 00–100 s, and high expression of genes Bo9g126150 and Bo9g111490 against both isolates in the resistant-line SCNU-98 indicate their putative roles in blackleg resistance, which remained to be functionally verified. This work enhances our understanding of R-gene-mediated resistance to blackleg in cabbage.

In silico characterization and expression of disease-resistance-related genes within the collinear region of Brassica napus blackleg resistant locus LepR1′ in B. oleracea

Blackleg is a devastating disease of cabbage (C genome), but so far, only a few resistant cabbage lines have been identified, and the resistance-related genes are yet to be identified. In contrast, numerous R loci have been mapped to the A or B genome. High ancestral synteny among Brassicaceae genomes suggest that homologous regions of R loci in the A or B genome may contain functional R gene(s) in the C genome. Here, we investigated the collinear region of a major Brassica napus R locus, LepR1′, in Brassica oleracea to identify genes of putative disease resistance class via comprehensive in silico analysis. The LepR1′ locus is collinear to a 17.29-Mbp region in chromosome C02 of Brassica oleracea and hosted a total of 1,392 genes, and 36 of these genes contained NBS, LRR, TIR, F-box and RLK domains. The expression of these 36 genes was profiled using qRT-PCR in the cotyledons of resistant (SCNU-03) and susceptible (SCNU-59) lines at 0, 6, 24, and 48 h after inoculation with Leptosphaeria maculans isolates 00–100s and 03–02s having the corresponding avirulent gene, AvrLep1. Among these genes, a cluster of 10 genes were differentially expressed. NBS-LRR gene Bo2g131620 had a 45-bp insertion/deletion (indel) mutation in the 3rd intron between resistant and susceptible lines. The higher expression in resistant lines against both isolates and the indel mutation in Bo2g131620 suggest that these genes may have roles in blackleg resistance in cabbage. Mapping and functional analysis will be necessary to validate such a role. Saturating the region with enough markers is recommended during mapping.

Deployment of Genetic and Genomic Tools Toward Gaining a Better Understanding of Rice-Xanthomonas oryzae pv. oryzae Interactions for Development of Durable Bacterial Blight Resistant Rice.

Rice is the most important food crop worldwide and sustainable rice production is important for ensuring global food security. Biotic stresses limit rice production significantly and among them, bacterial blight (BB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo) is very important. BB reduces rice yields severely in the highly productive irrigated and rainfed lowland ecosystems and in recent years; the disease is spreading fast to other rice growing ecosystems as well. Being a vascular pathogen, Xoo interferes with a range of physiological and biochemical exchange processes in rice. The response of rice to Xoo involves specific interactions between resistance (R) genes of rice and avirulence (Avr) genes of Xoo, covering most of the resistance genes except the recessive ones. The genetic basis of resistance to BB in rice has been studied intensively, and at least 44 genes conferring resistance to BB have been identified, and many resistant rice cultivars and hybrids have been developed and released worldwide. However, the existence and emergence of new virulent isolates of Xoo in the realm of a rapidly changing climate necessitates identification of novel broad-spectrum resistance genes and intensification of gene-deployment strategies. This review discusses about the origin and occurrence of BB in rice, interactions between Xoo and rice, the important roles of resistance genes in plant’s defense response, the contribution of rice resistance genes toward development of disease resistance varieties, identification and characterization of novel, and broad-spectrum BB resistance genes from wild species of Oryza and also presents a perspective on potential strategies to achieve the goal of sustainable disease management.

Identification of resistance loci in Chinese and Canadian canola/rapeseed varieties against Leptosphaeria maculans based on genome-wide association studies

BackgroundThe fungal pathogen Leptosphaeria maculans (Lm). causes blackleg disease on canola/rapeseed in many parts of the world. It is important to use resistant cultivars to manage the disease and minimize yield losses. In this study, twenty-two Lm isolates were used to identify resistance genes in a collection of 243 canola/rapeseed (Brassica napus L.) accessions from Canada and China. These Lm isolates carry different compliments of avirulence genes, and the investigation was based on a genome-wide association study (GWAS) and genotype-by-sequencing (GBS).ResultsUsing the CROP-SNP pipeline, a total of 81,471 variants, including 78,632 SNPs and 2839 InDels, were identified. The GWAS was performed using TASSEL 5.0 with GLM + Q model. Thirty-two and 13 SNPs were identified from the Canadian and Chinese accessions, respectively, tightly associated with blackleg resistance with P values < 1 × 10− 4. These SNP loci were distributed on chromosomes A03, A05, A08, A09, C01, C04, C05, and C07, with the majority of them on A08 followed by A09 and A03. The significant SNPs identified on A08 were all located in a 2010-kb region and associated with resistance to 12 of the 22 Lm isolates. Furthermore, 25 resistance gene analogues (RGAs) were identified in these regions, including two nucleotide binding site (NBS) domain proteins, fourteen RLKs, three RLPs and six TM-CCs. These RGAs can be the potential candidate genes for blackleg resistance.ConclusionThis study provides insights into potentially new genomic regions for discovery of additional blackleg resistance genes. The identified regions associated with blackleg resistance in the germplasm collection may also contribute directly to the development of canola varieties with novel resistance genes against blackleg of canola.

Identification of Natural Resistance Mediated by Recognition of Phytophthora infestans Effector Gene Avr3aEM in Potato

Late blight is considered the most renowned devastating potato disease worldwide. Resistance gene (R)-based resistance to late blight is the most effective method to inhibit infection by the causal agent Phytophthora infestans. However, the limited availability of resistant potato varieties and the rapid loss of R resistance, caused by P. infestans virulence variability, make disease control rely on fungicide application. We employed an Agrobacterium tumefaciens-mediated transient gene expression assay and effector biology approach to understand late blight resistance of Chinese varieties that showed years of promising field performance. We are particularly interested in PiAvr3aEM, the most common virulent allele of PiAvr3aKI that triggers a R3a-mediated hypersensitive response (HR) and late blight resistance. Through our significantly improved A. tumefaciens-mediated transient gene expression assay in potato using cultured seedlings, we characterized two dominant potato varieties, Qingshu9 and Longshu7, in China by transient expression of P. infestans effector genes. Transient expression of 10 known avirulence genes showed that PiAvr4 and PiAvr8 (PiAvrsmira2) could induce HR in Qingshu9, and PiAvrvnt1.1 in Longshu7, respectively. Our study also indicated that PiAvr3aEM is recognized by these two potato varieties, and is likely involved in their significant field performance of late blight resistance. The identification of natural resistance mediated by PiAvr3aEM recognition in Qingshu9 and Longshu7 will facilitate breeding for improved potato resistance against P. infestans.

An Avirulence Gene Cluster in the Wheat Stripe Rust Pathogen (Puccinia striiformis f. sp. tritici) Identified through Genetic Mapping and Whole-Genome Sequencing of a Sexual Population.

Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe (yellow) rust, is an obligate, biotrophic fungus. It was difficult to study the genetics of the pathogen due to the lack of sexual reproduction. The recent discovery of alternate hosts for P. striiformis f. sp. tritici makes it possible to study inheritance and map genes involved in its interaction with plant hosts. To identify avirulence (Avr) genes in P. striiformis f. sp. tritici, we developed a segregating population by selfing isolate 12-368 on barberry (Berberis vulgaris) plants under controlled conditions. The dikaryotic sexual population segregated for avirulent/virulent phenotypes on nine Yr single-gene lines. The parental and progeny isolates were whole-genome sequenced at >30× coverage using Illumina HiSeq PE150 technology. A total of 2,637 high-quality markers were discovered by mapping the whole-genome sequencing (WGS) reads to the reference genome of strain 93-210 and used to construct a genetic map, consisting of 41 linkage groups, spanning 7,715.0 centimorgans (cM) and covering 68 Mb of the reference genome. The recombination rate was estimated to be 1.81 ± 2.32 cM/10 kb. Quantitative trait locus analysis mapped six Avr gene loci to the genetic map, including an Avr cluster harboring four Avr genes, AvYr7, AvYr43, AvYr44, and AvYrExp2 Aligning the genetic map to the reference genome identified Avr candidates and narrowed them to a small genomic region (<200 kb). The discovery of the Avr gene cluster is useful for understanding pathogen evolution, and the identification of candidate genes is an important step toward cloning Avr genes for studying molecular mechanisms of pathogen-host interactions.IMPORTANCE Stripe rust is a destructive disease of wheat worldwide. Growing resistant cultivars is the most effective, easy-to-use, economical, and environmentally friendly strategy for the control of the disease. However, P. striiformis f. sp. tritici can produce new virulent races that may circumvent race-specific resistance. Therefore, understanding the genetic basis of the interactions between wheat genes for resistance and P. striiformis f. sp. tritici genes for avirulence is useful for improving cultivar resistance for more effective control of the disease. This study developed a high-quality map that facilitates genomic and genetic studies of important traits related to pathogen pathogenicity and adaptation to different environments and crop cultivars carrying different resistance genes. The information on avirulence/virulence genes identified in this study can be used for guiding breeding programs to select combinations of genes for developing new cultivars with effective resistance to mitigate this devastating disease.

Effector Repertoire of Phytophthora betacei: In Search of Possible Virulence Factors Responsible for Its Host Specificity.

Phytophthora betacei is an oomycete plant pathogen closely related to Phytophthora infestans. It infects tree tomato (Solanum betaceum) in northern South America, but is, under natural conditions, unable to infect potatoes or tomatoes, the main hosts of its sister species P. infestans. We characterized, and compared the effector repertoires of P. betacei and other Phytophthora species. To this end, we used in silico approaches to predict and describe the repertoire of secreted proteins in Phytophthora species and determine unique and core effectors. P. betacei has the largest proteome and secretome of all Phytophthora species evaluated. We identified between 450 and 1933 candidate effector genes in Phytophthora ramorum, Phytophthora sojae, Phytophthora cactorum, Phytophthora parasitica, Phytophthora palmivora, P. infestans, and P. betacei genomes. The P. betacei predicted secretome contains 5653 proteins, 1126 of which are apoplastic effectors and 807cytoplasmic effectors. Genes encoding cytoplasmic effectors include 791 genes with an RxLR domain (the largest number known so far in a Phytophthora species) and 16 with a Crinkler (CRN) domain. We detected homologs of previously described avirulence gene (Avr) present in Phytophthora spp., such as Avr1, Avr3b, Avr4, and Avrblb1, suggesting a high level of effector gene conservation among Phytophthora species. Nonetheless, fewer CRN effectors were obtained in P. betacei compared to all other Phytophthora species analyzed. The comparison between P. infestans and P. betacei effector profiles shows unique features in P. betacei that might be involved in pathogenesis and host preference. Indeed, 402 unique predicted effector genes were detected in P. betacei, corresponding to 197 apoplastic effector genes, 203 RxLR cytoplasmic effector genes, and 2 effector genes with CRN domain. This is the first characterization of the effector profile of P. betacei and the broadest comparison of predicted effector repertoires in the genus Phytophthora following a standardized prediction pipeline. The resultant P. betacei putative effector repertoire provides a reasonable set of proteins whose experimental validation could lead to understand the specific virulence factors responsible for the host specificity of this species.

Characterisation of the Pseudomonas savastanoi pv. phaseolicola population found in Eastern Australia associated with halo blight disease in Vigna radiata

This study analysed the phenotypic and genotypic variation among 511 Pseudomonas savastanoi pv. phaseolicola (Psp) isolates, causing halo blight in mungbeans. Collected from symptomatic mungbean (Vigna radiata) crops throughout Australia between 2005 and 2018, a total of 352 Psp isolates were phenotypically screened. Our in planta screening against a set of four mungbean cultivars with known susceptible and resistant reactions revealed five distinctive pathotypes. Isolates belonging to pathotype 2 were the most prevalent at 84% and were found to be highly pathogenic towards all tested mungbean genotypes. Genomic variation was investigated for 205 isolates using DNA fingerprints, splitting the halo blight pathogen population into two broad genetic lineages. Further genetic testing for two known avirulence genes, avrPphE and avrPphF, identified the avrPphE gene in all the tested isolates and avrPphF present in all but two. To identify candidate avirulence genes unique to Psp isolates infecting mungbean in Australia, a comparative genomics study was undertaken on the whole-genome sequences of two epidemiologically important Psp isolates, T11544 and K4287. The information presented in this study has the potential to dramatically improve mungbean disease resistance now and into the future.

Long-Read-Based de novo Genome Assembly and Comparative Genomics of the Wheat Leaf Rust Pathogen Puccinia triticina Identifies Candidates for Three Avirulence Genes.

Leaf rust, caused by Puccinia triticina (Pt), is one of the most devastating diseases of wheat, affecting production in nearly all wheat-growing regions worldwide. Despite its economic importance, genomic resources for Pt are very limited. In the present study, we have used long-read sequencing (LRS) and the pipeline of FALCON and FALCON-Unzip (v4.1.0) to carry out the first LRS-based de novo genome assembly for Pt. Using 22.4-Gb data with an average read length of 11.6 kb and average coverage of 150-fold, we generated a genome assembly for Pt104 [strain 104-2,3,(6),(7),11; isolate S423], considered to be the founding isolate of a clonal lineage of Pt in Australia. The Pt104 genome contains 162 contigs with a total length of 140.5 Mb and N50 of 2 Mb, with the associated haplotigs providing haplotype information for 91% of the genome. This represents the best quality of Pt genome assembly to date, which reduces the contig number by 91-fold and improves the N50 by 4-fold as compared to the previous Pt race1 assembly. An annotation pipeline that combined multiple lines of evidence including the transcriptome assemblies derived from RNA-Seq, previously identified expressed sequence tags and Pt race 1 protein sequences predicted 29,043 genes for Pt104 genome. Based on the presence of a signal peptide, no transmembrane segment, and no target location to mitochondria, 2,178 genes were identified as secreted proteins (SPs). Whole-genome sequencing (Illumina paired-end) was performed for Pt104 and six additional strains with differential virulence profile on the wheat leaf rust resistance genes Lr26, Lr2a, and Lr3ka. To identify candidates for the corresponding avirulence genes AvrLr26, AvrLr2a, and AvrLr3ka, genetic variation within each strain was first identified by mapping to the Pt104 genome. Variants within predicted SP genes between the strains were then correlated to the virulence profiles, identifying 38, 31, and 37 candidates for AvrLr26, AvrLr2a, and AvrLr3ka, respectively. The identification of these candidate genes lays a good foundation for future studies on isolating these avirulence genes, investigating the molecular mechanisms underlying host–pathogen interactions, and the development of new diagnostic tools for pathogen monitoring.

High nucleotide sequence variation of avirulent gene, AVR-Pita1, in Thai rice blast fungus population

Rice blast disease, caused by Magnaporthe oryzae, is one of the most importance diseases of rice production worldwide. The keyrole of defense mechanism to combat this fungus in rice follows the gene-for-gene concept, which a plant resistant (R) gene product recognizes a fungal avirulent (AVR) effector and triggers the hypersensitive response. However, the AVR genes have been shown to be rapidly evolving resulting in high level of genetic diversity. The aims of this study were to examine the nucleotide sequence variation of AVR-Pita1 gene in Thai rice blast isolates and to identify the severity of blast disease using isogenic line of Pita gene. Seventy-six rice blast isolates collected from different parts of Thailand were used. Gene specific primers for AVR-Pita1 gene coding sequence were designed and used for identifying the genetic diversity of AVR-Pita1 gene by PCR amplification and sequencing. The obtained sequences were analysed for genetic variation and genetic relationship. Our results revealed the association between the sequence variations of AVR-Pita1 and selective forces from Pita gene. This phenomenon demonstrated the coevolution between rice blast resistant gene in rice and avirulent gene in blast fungus. The information about variation and evolutionary mechanisms of AVR gene obtained from this study can be used in rice blast resistant breeding programme.