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The soybean–Phytophthora sojae interaction operates on a gene‐for‐gene relationship, where the product of a resistance gene (Rps) in the host recognizes that of an avirulence gene (Avr) in the pathogen to generate an incompatible reaction. To exploit this form of resistance, one must match with precision the appropriate Rps gene with the corresponding Avr gene. Currently, this association is evaluated by phenotyping assays that are labour‐intensive and often imprecise. To circumvent this limitation, we sought to develop a molecular assay that would reveal the avirulence allele of the seven main Avr genes (Avr1a, Avr1b, Avr1c, Avr1d, Avr1k, Avr3a, and Avr6) in order to diagnose with precision the pathotypes of P. sojae isolates. For this purpose, we analysed the genomic regions of these Avr genes in 31 recently sequenced isolates with different virulence profiles and identified discriminant mutations between avirulence and virulence alleles. Specific primers were designed to generate amplicons of a distinct size, and polymerase chain reaction conditions were optimized in a final assay of two parallel runs. When tested on the 31 isolates of known virulence, the assay accurately revealed all avirulence alleles. The test was further assessed and compared to a phenotyping assay on 25 isolates of unknown virulence. The two assays matched in 97% (170/175) of the interactions studied. Interestingly, the sole cases of discrepancy were obtained with Avr3a, which suggests a possible imperfect interaction with Rps3a. This molecular assay offers a powerful and reliable tool to exploit and study with greater precision soybean resistance against P. sojae. This study describes a molecular assay capable of defining the pathotypes of P. sojae and offers a powerful and reliable tool to exploit and study with greater precision soybean resistance against P. sojae.

Broad-spectrum resistance has great values for crop breeding. However, its mechanisms are largely unknown. Here, we report the cloning of a maize NLR gene, RppK , for resistance against southern corn rust (SCR) and its cognate Avr gene, AvrRppK , from Puccinia polysora (the causal pathogen of SCR). The AvrRppK gene has no sequence variation in all examined isolates. It has high expression level during infection and can suppress pattern-triggered immunity (PTI). Further, the introgression of RppK into maize inbred lines and hybrids enhances resistance against multiple isolates of P. polysora , thereby increasing yield in the presence of SCR. Together, we show that RppK is involved in resistance against multiple P. polysora isolates and it can recognize AvrRppK, which is broadly distributed and conserved in P. polysora isolates. Southern corn rust (SCR) caused by Puccinia polysora is a major maize disease that can result in major yield loss. Here, the authors report the expression of a CC-NB-LRR type of R gene RppK results in SCR resistance in susceptible maize lines and it can recognize the effector AvrRppK produced by P. polysora .

Recognition of a pathogen avirulence (AVR) effector protein by a cognate plant resistance (R) protein triggers a set of immune responses that render the plant resistant. Pathogens can escape this so-called Effector-Triggered Immunity (ETI) by different mechanisms including the deletion or loss-of-function mutation of the AVR gene, the incorporation of point mutations that allow recognition to be evaded while maintaining virulence function, and the acquisition of new effectors that suppress AVR recognition. The Dothideomycete Leptosphaeria maculans , causal agent of oilseed rape stem canker, is one of the few fungal pathogens where suppression of ETI by an AVR effector has been demonstrated. Indeed, AvrLm4-7 suppresses Rlm3- and Rlm9-mediated resistance triggered by AvrLm3 and AvrLm5-9, respectively. The presence of AvrLm4-7 does not impede AvrLm3 and AvrLm5-9 expression, and the three AVR proteins do not appear to physically interact. To decipher the epistatic interaction between these L . maculans AVR effectors, we determined the crystal structure of AvrLm5-9 and obtained a 3D model of AvrLm3, based on the crystal structure of Ecp11-1, a homologous AVR effector candidate from Fulvia fulva . Despite a lack of sequence similarity, AvrLm5-9 and AvrLm3 are structural analogues of AvrLm4-7 (structure previously characterized). Structure-informed sequence database searches identified a larger number of putative structural analogues among L . maculans effector candidates, including the AVR effector AvrLmS-Lep2, all produced during the early stages of oilseed rape infection, as well as among effector candidates from other phytopathogenic fungi. These structural analogues are named LARS (for Leptosphaeria AviRulence and Suppressing) effectors. Remarkably, transformants of L . maculans expressing one of these structural analogues, Ecp11-1, triggered oilseed rape immunity in several genotypes carrying Rlm3 . Furthermore, this resistance could be suppressed by AvrLm4-7. These results suggest that Ecp11-1 shares a common activity with AvrLm3 within the host plant which is detected by Rlm3, or that the Ecp11-1 structure is sufficiently close to that of AvrLm3 to be recognized by Rlm3.

During infection, pathogens secrete an arsenal of molecules, collectively called effectors, key elements of pathogenesis which modulate innate immunity of the plant and facilitate infection. Some of these effectors can be recognized directly or indirectly by resistance (R) proteins from the plant and are then called avirulence (AVR) proteins. This recognition usually triggers defense responses including the hypersensitive response and results in resistance of the plant. - gene interactions are frequently exploited in the field to control diseases. Recently, the availability of fungal genomes has accelerated the identification of genes in plant pathogenic fungi, including in fungi infecting agronomically important crops. While single genes recognized by their corresponding gene were identified, more and more complex interactions between and genes are reported (e.g., genes recognized by several genes, genes recognizing several genes in distinct organisms, one gene suppressing recognition of another gene by its corresponding gene, two cooperating genes both necessary to recognize an gene). These complex interactions were particularly reported in pathosystems showing a long co-evolution with their host plant but could also result from the way agronomic crops were obtained and improved (e.g., through interspecific hybridization or introgression of resistance genes from wild related species into cultivated crops). In this review, we describe some complex interactions between plants and fungi that were recently reported and discuss their implications for gene evolution and gene management.

This article is a Commentary on Praz et al., 213: 1301–1314.

Background Rice blast disease is one of the most destructive fungal disease of rice worldwide. The avirulence ( AVR ) genes of Magnaporthe oryzae are recognized by the cognate resistance ( R ) genes of rice and trigger race-specific resistance. The variation in AVR is one of the major drivers of new races. Detecting the variation in the AVR gene in isolates from a population of Magnaporthe oryzae collected from rice production fields will aid in evaluating the effectiveness of R genes in rice production areas. The Pik gene contains 5 R alleles ( Pik , Pikh , Pikp , Pikm and Piks ) corresponding to the AVR alleles ( AVR-Pik/kh/kp/km/ks ) of M. oryzae . The Pik gene specifically recognizes and prevents infections by isolates of M. oryzae that contain AVR-Pik . The molecular variation in AVR - Pik alleles of M. oryzae and Pik alleles of rice remains unclear. Results We studied the possible evolutionary pathways of AVR-Pik alleles by analyzing their DNA sequence variation and assaying their avirulence to the cognate Pik alleles of resistance genes under field conditions in China. The results of PCR products from genomic DNA showed that 278 of the 366 isolates of M. oryzae collected from Yunnan Province, China, carried AVR-Pik alleles. Among the isolates from six regions of Yunnan, 66.7–90.3% carried AVR-Pik alleles. Moreover, 10 AVR-Pik haplotypes encoding five novel AVR-Pik variants were identified among 201 isolates. The AVR-Pik alleles evolved to virulent from avirulent forms via stepwise base substitution. These findings demonstrate that AVR-Pik alleles are under positive selection and that mutations are responsible for defeating race-specific resistant Pik alleles in nature. Conclusions We demonstrated the polymorphism and distribution of AVR - Pik alleles in Yunnan Province, China. By pathogenicity assays used to detect the function of the different haplotypes of AVR - Pik , for the first time, we showed the avoidance and stepwise evolution of AVR - Pik alleles in rice production areas of Yunnan. The functional AVR-Pik possesses diversified sequence structures and is under positive selection in nature.

The use of resistance genes in elite soybean cultivars is one of the most widely used methods to manage Phytophthora sojae. This method relies on effector‐triggered immunity, where a Resistant to P. sojae (Rps) gene product from the plant recognizes a specific effector from the pathogen, encoded by an avirulence (Avr) gene. Many Avr genes from P. sojae have been identified in the last decade, allowing a better exploitation of this type of resistance. The objective of the present study was to identify the Avr gene triggering immunity derived from the soybean resistance gene Rps8. The analysis of a segregating F2 progeny coupled with a genotyping‐by‐sequencing approach led to the identification of a putative Avr8 locus. The investigation of this locus using whole‐genome sequencing data from 31 isolates of P. sojae identified Avr3a as the likely candidate for Avr8. Long‐read sequencing also revealed that P. sojae isolates can carry up to five copies of the Avr3a gene, compared to the four previously reported. Haplotype and transcriptional analyses showed that amino acid changes and absence of Avr3a transcripts from P. sojae isolates caused changes in virulence towards Rps8. Functional analyses using CRISPR/Cas9 knockout and constitutive expression demonstrated that Rps8 interacted with Avr3a. We also showed that a specific allele of Avr3a is recognized by Rps3a but not Rps8. While Rps3a and Rps8 have been previously described as closely linked, this is the first report of a clear distinction hitherto undefined between these two resistance genes. The RXLR effector coded by a specific allele of the avirulence gene Avr3a from Phytophthora sojae (causing Phytophthora root rot) triggers immunity derived from the soybean resistance gene Rps8.

In the plant immune system, according to the ‘gene-for-gene’ model, a resistance (R) gene product in the plant specifically surveils a corresponding effector protein functioning as an avirulence (Avr) gene product. This system differs from other plant–pathogen interaction systems, in which plant R genes recognize a single type of gene or gene family because almost all virus genes with distinct structures and functions can also interact with R genes as Avr determinants. Thus, research conducted on viral Avr-R systems can provide a novel understanding of Avr and R gene product interactions and identify mechanisms that enable rapid co-evolution of plants and phytopathogens. In this review, we intend to provide a brief overview of virus-encoded proteins and their roles in triggering plant resistance, and we also summarize current progress in understanding plant resistance against virus Avr genes. Moreover, we present applications of Avr gene-mediated phenotyping in R gene identification and screening of segregating populations during breeding processes.

Wheat stem rust caused by the fungus Puccinia graminis f. sp. tritici ( Pgt ), is regaining prominence due to the recent emergence of virulent isolates and epidemics in Africa, Europe and Central Asia. The development and deployment of wheat cultivars with multiple stem rust resistance ( Sr ) genes stacked together will provide durable resistance. However, certain disease resistance genes can suppress each other or fail in particular genetic backgrounds. Therefore, the function of each Sr gene must be confirmed after incorporation into an Sr -gene stack. This is difficult when using pathogen disease assays due to epistasis from recognition of multiple avirulence (Avr) effectors. Heterologous delivery of single Avr effectors can circumvent this limitation, but this strategy is currently limited by the paucity of cloned Pgt Avrs . To accelerate Avr gene cloning, we outline a procedure to develop a mutant population of Pgt spores and select for gain-of-virulence mutants. We used ethyl methanesulphonate (EMS) to mutagenize urediniospores and create a library of > 10,000 independent mutant isolates that were combined into 16 bulks of ~658 pustules each. We sequenced random mutants and determined the average mutation density to be 1 single nucleotide variant (SNV) per 258 kb. From this, we calculated that a minimum of three independently derived gain-of-virulence mutants is required to identify a given Avr gene. We inoculated the mutant library onto plants containing Sr43 , Sr44 , or Sr45 and obtained 9, 4, and 14 mutants with virulence toward Sr43 , Sr44 , or Sr45 , respectively. However, only mutants identified on Sr43 and Sr45 maintained their virulence when reinolculated onto the lines from which they were identified. We further characterized 8 mutants with virulence toward Sr43 . These also maintained their virulence profile on the stem rust international differential set containing 20 Sr genes, indicating that they were most likely not accidental contaminants. In conclusion, our method allows selecting for virulent mutants toward targeted resistance ( R ) genes. The development of a mutant library from as little as 320 mg spores creates a resource that enables screening against several R genes without the need for multiple rounds of spore multiplication and mutagenesis.

Crop breeding for durable disease resistance is challenging due to the rapid evolution of pathogen virulence. While progress in resistance (R) gene cloning and stacking has accelerated in recent years1–3, the identification of corresponding avirulence (Avr) genes in many pathogens is hampered by the lack of high-throughput screening options. To address this technology gap, we developed a platform for pooled library screening in plant protoplasts to allow rapid identification of interacting R–Avr pairs. We validated this platform by isolating known and novel Avr genes from wheat stem rust (Puccinia graminis f. sp. tritici) after screening a designed library of putative effectors against individual R genes. Rapid Avr gene identification provides molecular tools to understand and track pathogen virulence evolution via genotype surveillance, which in turn will lead to optimized R gene stacking and deployment strategies. This platform should be broadly applicable to many crop pathogens and could potentially be adapted for screening genes involved in other protoplast-selectable traits.The authors developed a platform for rapid identification of interacting plant immune receptors and pathogen avirulence proteins by library screening in protoplasts, then used it to identify new wheat stem rust Avr genes recognized by known wheat resistance genes.