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Barley net form net blotch (NFNB) is a destructive foliar disease caused by Pyrenophora teres f. teres. Barley line CIho5791, which harbors the broadly effective chromosome 6H resistance gene Rpt5, displays dominant resistance to P. teres f. teres. To genetically characterize P. teres f. teres avirulence/virulence on the barley line CIho5791, we generated a P. teres f. teres mapping population using a cross between the Moroccan CIho5791-virulent isolate MorSM40-3 and the avirulent reference isolate 0-1. Full genome sequences were generated for 103 progenies. Saturated chromosome-level genetic maps were generated, and quantitative trait locus (QTL) mapping identified two major QTL associated with P. teres f. teres avirulence/virulence on CIho5791. The most significant QTL mapped to chromosome (Ch) 1, where the virulent allele was contributed by MorSM40-3. A second QTL mapped to Ch8; however, this virulent allele was contributed by the avirulent parent 0-1. The Ch1 and Ch8 loci accounted for 27 and 15% of the disease variation, respectively, and the avirulent allele at the Ch1 locus was epistatic over the virulent allele at the Ch8 locus. As a validation, we used a natural P. teres f. teres population in a genome-wide association study that identified the same Ch1 and Ch8 loci. We then generated a new reference quality genome assembly of parental isolate MorSM40-3 with annotation supported by deep transcriptome sequencing of infection time points. The annotation identified candidate genes predicted to encode small, secreted proteins, one or more of which are likely responsible for overcoming the CIho5791 resistance. [Figure: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

The wheat fungal pathogens Puccinia graminis f. sp. tritici (Pgt), P. striiformis f. sp. tritici, and P. triticina, causing stem, stripe, and leaf rust, respectively, pose a threat to global wheat production. Genetic resistance in the form of resistance (R) genes provides the best protection, but rust fungal populations change frequently by mutating avirulence (AVR) effectors matching specific R genes, thereby defeating resistance. Hence, characterization of AVR effectors is needed to understand the evolution of pathogen populations, provide insights for extending the effectiveness of R genes, and yield tools for the identification and isolation of R genes. Functional characterization of Avr genes in rust fungi is challenging in these biotrophic pathogens that lack a reliable and efficient transformation system. Studies indicate that the recently engineered foxtail mosaic virus (FoMV) shows promise as an expression system in cereals. In this study, we utilized two confirmed AVR effectors from Pgt, AVRSr35 and AVRSr50, to assess the applicability of FoMV for investigating rust fungus Avr genes. We showed that vector FoMV PV101 carrying PgtAvrSr35 induced a hypersensitive response (HR) in wheat having the corresponding Sr35 resistance gene. However, when carrying PgtAvrSr50, no HR or even mild viral symptoms were seen in a wheat line having Sr50, as this particular wheat line was not susceptible to FoMV. Several wheat cultivars did not support FoMV replication. The results here show that FoMV PV101 is effective as an expression vector for studying rust fungi AVR effectors, but its applicability relies on the susceptibility of wheat cultivars to FoMV. [Figure: see text] Copyright © 2024 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada. This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Crops are constantly exposed to pathogenic microbes. Rust fungi are examples of these harmful microorganisms, which have a major economic impact on wheat production. To protect themselves from pathogens like rust fungi, plants employ a multilayered immune system that includes immunoreceptors encoded by resistance genes. Significant efforts have led to the isolation of numerous resistance genes against rust fungi in cereals, especially in wheat. However, the evolution of virulence of rust fungi hinders the durability of resistance genes as a strategy for crop protection. Rust fungi, like other biotrophic pathogens, secrete an arsenal of effectors to facilitate infection, and these are the molecules that plant immunoreceptors target for pathogen recognition and mounting defense responses. When recognized, these effector proteins are referred to as avirulence (Avr) effectors. Despite the many predicted effectors in wheat rust fungi, only five Avr genes have been identified, all from wheat stem rust. Knowledge of the Avr genes and their variation in the fungal population will inform deployment of the most appropriate wheat disease-resistance genes for breeding and farming. The review provides an overview of methodologies as well as the validation techniques that have been used to characterize Avr effectors from wheat stem rust. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The gene-for-gene model proposed by H. H. Flor has been one of the fundamental precepts of plant-pathogen interactions that has underpinned decades of research towards our current concepts of plant immunity. The broad validity of this model as an elegant and accurate genetic description of specific recognition events between the products of plant resistance (R) and pathogen avirulence (Avr) genes has been demonstrated many times over in a wide variety of plant disease systems. In recent years detailed molecular and structural analyses have provided a deep understanding of the principles by which plant immune receptors recognize pathogen effectors, including providing molecular descriptions of many of the genetic loci in flax and flax rust characterized by Flor. Recent advances in molecular and structural understanding of immune receptor recognition and activation mechanisms have brought the field to a new level, where rational design of novel receptors through engineering approaches is becoming a realizable goal. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Ralstonia solanacearum causes bacterial wilt disease in solanaceous crops. Identification of avirulence type III-secreted effectors recognized by specific disease resistance proteins in host plant species is an important step toward developing durable resistance in crops. In the present study, we show that R. solanacearum effector RipJ functions as an avirulence determinant in Solanum pimpinellifolium LA2093. In all, 10 candidate avirulence effectors were shortlisted based on the effector repertoire comparison between avirulent Pe_9 and virulent Pe_1 strains. Infection assays with transgenic strain Pe_1 individually carrying a candidate avirulence effector from Pe_9 revealed that only RipJ elicits strong bacterial wilt resistance in S. pimpinellifolium LA2093. Furthermore, we identified that several RipJ natural variants do not induce bacterial wilt resistance in S. pimpinellifolium LA2093. RipJ belongs to the YopJ family of acetyltransferases. Our sequence analysis indicated the presence of partially conserved putative catalytic residues. Interestingly, the conserved amino acid residues in the acetyltransferase catalytic triad are not required for effector-triggered immunity. In addition, we show that RipJ does not autoacetylate its lysine residues. Our study reports the identification of the first R. solanacearum avirulence protein that triggers bacterial wilt resistance in tomato. We expect that our discovery of RipJ as an avirulence protein will accelerate the development of bacterial wilt-resistant tomato varieties in the future.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The Mi-1 gene in tomato confers effective resistance against several species of root-knot nematode, including Meloidogyne javanica. A strain of M. javanica that can reproduce on tomato with Mi-1 was obtained from a culture of an avirulent strain after greenhouse selection. DNA blots and amplified fragment length polymorphism (AFLP) analysis indicated that the two nematode strains are closely related. Expression patterns visualized as cDNA AFLPs were nearly identical except for a cDNA fragment, Cg-1, that was present in the avirulent strain but not in the virulent strain. DNA blots showed that Cg-1 corresponds to a member of a small gene family with one or more copies missing in the virulent strain compared with the avirulent strain. Except for the presence of a histone stem loop near the 3′ end of the transcript, Cg-1 shows no similarity to other sequences in GenBank. The longest open reading frame is 32 amino acids and initiates at the fourth AUG in the predicted transcript. When nematode juveniles of the Mi-1-avirulent strain were soaked in dsRNA corresponding to part of the predicted Cg-1 transcript, they produced progeny that were virulent on tomato carrying the Mi-1 gene, strongly suggesting that Cg-1 is required in the nematode for Mi-1-mediated resistance.

Effector proteins injected by the pathogenic bacteria Pseudomonas syringae into plants can have profound effects on the pathogen–host interaction due to their efficient recognition by plants and the subsequent triggering of defenses. The AvrRpt2 effector triggers strong local and systemic defense (called systemic acquired resistance [SAR]) responses in Arabidopsis thaliana plants that harbor a functional RPS2 gene that encodes an R protein in the coiled-coil, nucleotide-binding domain, leucine-rich repeat class. The newly identified win3-T mutant shows greatly reduced resistance to P. syringae carrying avrRpt2. In win3-T plants, RIN4 cleavage, an early AvrRpt2-induced event, is normal. However, salicylic acid accumulation is compromised, as is SAR induction and the local hypersensitive cell death response after infection by P. syringae carrying avrRpt2. WIN3 encodes a member of the firefly luciferase protein superfamily. Expression of WIN3 at an infection site partially requires PAD4, a protein known to play a quantitative role in RPS2-mediated signaling. WIN3 expression in tissue distal to an infection site requires multiple salicylic acid regulatory genes. Finally, win3-T plants show modestly increased susceptibility to virulent P. syringae and modestly reduced SAR in response to P. syringae carrying avrRpm1. Thus, WIN3 is a key element of the RPS2 defense response pathway and a basal and systemic defense component.

Xanthomonas campestris pv. vesicatoria bacteria expressing the type III effector protein AvrBs3 induce a hypersensitive response in pepper plants carrying the resistance gene Bs3. Here, we report that infection of susceptible pepper and tomato plants leads to an AvrBs3-dependent hypertrophy of the mesophyll tissue. Agrobacterium-mediated transient expression of the avrBs3 gene in tobacco and potato plants resulted in a similar phenotype. Induction of hypertrophy was shown to depend on the repeat region, nuclear localization signals, and acidic transcription activation domain (AAD) of AvrBs3, suggesting that the effector modulates the host's transcriptome. To search for host genes regulated by AvrBs3 in an AAD-dependent manner, we performed a cDNA-amplified fragment length polymorphism analysis of pepper mRNA populations. Thirteen AvrBs3-induced transcripts were identified and confirmed by reverse transcriptase-poly-merase chain reaction. Sequence analysis revealed homologies to auxin-induced and expansinlike genes, which play a role in cell enlargement. These results suggest that some of the AvrBs3-induced genes may be involved in hypertrophy development and that xanthomonads possess type III effectors that steer host gene expression.

Recently, the helicase domain of the Tobacco mosaic virus (TMV)-U1 replicase proteins (designated MOREHEL:U1) was identified as the elicitor of the N gene-mediated hypersensitive response (HR) in tobacco. In this study, we used agroinfiltration to express the equivalent MOREHEL domain of the non-HR-inducing tobamovirus strain TMV-Ob. It appeared that this MOREHEL:Ob sequence did not elicit a HR in N gene-carrying tobacco. Both MOREHEL sequences were divided into eight subdomains, and chimeras of MOREHEL sequences from U1 and Ob were constructed. Expression of these chimeric MOREHEL sequences revealed that, in the TMV-U1 MOREHEL sequence, at least four domains involved in full HR induction were present. The presence of at least three of these four domains seems a minimal requirement for HR induction. Two additional domains may play a minor role in HR induction. To study the elicitor function of the chimeras during the TMV life cycle, chimeric MOREHEL domains were introduced into full-length TMV cDNA clones. These constructs, however, were unable to establish an infection in Nicotiana benthamiana or Nicotiana tabacum plants.

The host range of the gall-forming bacterium Erwinia herbicola pv. gypsophilae (Ehg) is restricted to gypsophila whereas Erwinia herbicola pv. betae (Ehb) attacks beet as well as gypsophila. Both pathovars contain an indigenous plasmid (pPATHEhg or pPATHEhb) that harbors pathogenicity genes, including the hrp gene cluster. A cosmid library of Ehg824-1 plasmid DNA was mobilized into Ehb4188 and the transconjugants were screened for pathogenicity on beet. One Ehb transconjugant harboring the cosmid pLA173 of pPATHEhg induced a hypersensitive-like response and abolished pathogenicity on beet. Transposon mutagenesis of an open reading frame (ORF) located on this cosmid eliminated its affect on pathogenicity. Marker exchange of this mutation into Ehg824-1 caused a substantial reduction in gall size on gypsophila and caused Ehg824-1 to extend its host range and incite galls on beet. The ORF (1.5 kb) was designated as pthG (pathogenicity gene on gypsophila). DNA sequence analysis of pthG revealed no significant homology to known genes in the data bank. Only remnants of the pthG sequences were identified on the pPATH of Ehb4188. The deduced protein lacked an N-terminal signal peptide but contained a short trans-membrane helix in its C terminus. The gene product, as determined by expression in Escherichia coli and Western blots (immunoblots), was a 56-kDa protein.

A fundamental question about microbial pathogens is how they elicit host-specific symptoms. We report here that expression of a single gene from a plant-pathogenic bacterium in plant cells elicits host-specific symptoms diagnostic of the disease caused by the pathogen. Expression of pthA from Xanthomonas citri in citrus cells is sufficient to cause division, enlargement, and death of host cells. Since elicitation of these symptoms depends on a functional type III protein secretion system in X. citri, we deduce that the PthA protein is a specific plant signal, its site of action is inside the plant cell, and it is a major determinant of host range.

Induction of peroxidase has been correlated with resistant interactions between rice and Xanthomonas oryzae pv. oryzae. To assist in analysis of the role of rice peroxidases in plant defense against the bacterial pathogen, three peroxidase genes, POX22.3, POX8.1, and POX5.1, were identified from a rice cDNA library that was constructed from leaves of plants undergoing a resistant reaction. These genes were highly similar in nucleic acid and amino acid sequences and belonged to a gene family. The three genes showed differential expression in infiltrated rice leaves during pathogen interactions and mechanical stress. Only two peroxidase genes, POX8.1 and POX22.3, were predominantly expressed during resistant interactions. These two genes also were expressed during susceptible interactions, but induction was delayed compared with resistant interactions. POXgX9, a fourth peroxidase gene that was isolated from a genomic library, is adjacent to POX22.3 in the rice genome and has greater than 90% similarity in nucleotide and amino acid sequence identity to POX22.3. Interestingly, POXgX9 was expressed only in the roots of rice plants. While POX22.3 was expressed in both leaves and roots, POX8.1 and POX5.1 were not detected in roots but were induced in leaves by mechanical wounding at different times after treatment. POX22.3, POX8.1, and POX5.1 were estimated to be present in single copies in rice haploid genome. These results indicate that different members of the rice peroxidase gene family are distinctly regulated in response to various environmental cues.