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Opportunistic commensal and environmental fungi can cause superficial to systemic diseases in humans. But how did these pathogens adapt to infect us and how does host-pathogen coevolution shape their virulence potential? During evolution toward pathogenicity, not only do microorganisms gain virulence genes, but they also tend to lose non-adaptive genes in the host niche. Additionally, virulence factors can become detrimental during infection when they trigger host recognition. The loss of non-adaptive genes as well as the loss of the virulence potential of genes by adaptations to the host has been investigated in pathogenic bacteria and phytopathogenic fungi, where they are known as antivirulence and avirulence genes, respectively. However, these concepts are nearly unknown in the field of pathogenic fungi of humans. We think that this unnecessarily limits our view of human-fungal interplay, and that much could be learned if we applied a similar framework to aspects of these interactions. In this review, we, therefore, define and adapt the concepts of antivirulence and avirulence genes for human pathogenic fungi. We provide examples for analogies to antivirulence genes of bacterial pathogens and to avirulence genes of phytopathogenic fungi. Introducing these terms to the field of pathogenic fungi of humans can help to better comprehend the emergence and evolution of fungal virulence and disease. [ABSTRACT FROM AUTHOR]

Magnaporthe oryzae infects rice, wheat, and many grass species in the Poaceae family by secreting protein effectors. Here, we analyzed the distribution, sequence variation, and genomic context of effector candidate (EFC) genes in 31 isolates that represent five pathotypes of M. oryzae, three isolates of M. grisea, a sister species of M. oryzae, and one strain each for eight species in the family Magnaporthaceae to investigate how the host range expansion of M. oryzae has likely affected the evolution of effectors. We used the EFC genes of M. oryzae strain 70-15, whose genome has served as a reference for many comparative genomics analyses, to identify their homologs in these strains. We also analyzed the previously characterized avirulence (AVR) genes and single-copy orthologous (SCO) genes in these strains, which showed that the EFC and AVR genes evolved faster than the SCO genes. The EFC and AVR repertoires among M. oryzae pathotypes varied widely probably because adaptation to individual hosts exerted different types of selection pressure. Repetitive DNA elements appeared to have caused the variation of some EFC genes. Lastly, we analyzed expression patterns of the AVR and EFC genes to test the hypothesis that such genes are preferentially expressed during host infection. This comprehensive dataset serves as a foundation for future studies on the genetic basis of the evolution and host specialization in M. oryzae.

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. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Net form net blotch (NFNB), caused by Pyrenophora teres f. teres , is an important barley disease. The centromeric region of barley chromosome 6H has often been associated with resistance or susceptibility to NFNB, including the broadly effective dominant resistance gene Rpt5 derived from barley line CIho 5791. We characterized a population of Moroccan P. teres f. teres isolates that had overcome Rpt5 resistance and identified quantitative trait loci (QTL) that were effective against these isolates. Eight Moroccan P. teres f. teres isolates were phenotyped on barley lines CIho 5791 and Tifang. Six isolates were virulent on CIho 5791, and two were avirulent. A CIho 5791 × Tifang recombinant inbred line (RIL) population was phenotyped with all eight isolates and confirmed the defeat of the 6H resistance locus formerly mapped as Rpt5 in barley line CI9819. A major QTL on chromosome 3H with the resistance allele derived from Tifang, as well as minor QTL, was identified and provided resistance against these isolates. F 2 segregation ratios supported dominant inheritance for both the 3H and 6H resistance. Furthermore, inoculation of progeny isolates derived from a cross of P. teres f. teres isolates 0-1 (virulent on Tifang/avirulent on CIho 5791) and MorSM 40-3 (avirulent on Tifang/virulent on CIho 5791) onto the RIL and F 2 populations determined that recombination between isolates can generate novel genotypes that overcome both resistance genes. Markers linked to the QTL identified in this study can be used to incorporate both resistance loci into elite barley cultivars for durable resistance., Competing Interests: The author(s) declare no conflict of interest.

Pathogens and plants are in a constant battle with one another, the result of which is either the restriction of pathogen growth via constitutive or induced plant defense responses or the pathogen colonization of plant cells and tissues that cause disease. Elicitins are a group of highly conserved proteins produced by certain oomycete species, and their sterol binding ability is recognized as an important feature in sterol–auxotrophic oomycetes. Elicitins also orchestrate other aspects of the interactions of oomycetes with their plant hosts. The function of elicitins as avirulence or virulence factors is controversial and is dependent on the host species, and despite several decades of research, the function of these proteins remains elusive. We summarize here our current understanding of elicitins as either defense-promoting or defense-suppressing agents and propose that more recent approaches such as the use of ‘omics’ and gene editing can be used to unravel the role of elicitins in host–pathogen interactions. A better understanding of the role of elicitins is required and deciphering their role in host–pathogen interactions will expand the strategies that can be adopted to improve disease resistance and reduce crop losses.

Magnaporthe oryzae infects rice, wheat, and many grass species in the Poaceae family by secreting protein effectors. Here, we analyzed the distribution, sequence variation, and genomic context of effector candidate (EFC) genes in 31 isolates that represent five pathotypes of M. oryzae , three isolates of M. grisea , a sister species of M. oryzae , and one strain each for eight species in the family Magnaporthaceae to investigate how the host range expansion of M. oryzae has likely affected the evolution of effectors. We used the EFC genes of M. oryzae strain 70-15, whose genome has served as a reference for many comparative genomics analyses, to identify their homologs in these strains. We also analyzed the previously characterized avirulence (AVR) genes and single-copy orthologous (SCO) genes in these strains, which showed that the EFC and AVR genes evolved faster than the SCO genes. The EFC and AVR repertoires among M. oryzae pathotypes varied widely probably because adaptation to individual hosts exerted different types of selection pressure. Repetitive DNA elements appeared to have caused the variation of some EFC genes. Lastly, we analyzed expression patterns of the AVR and EFC genes to test the hypothesis that such genes are preferentially expressed during host infection. This comprehensive dataset serves as a foundation for future studies on the genetic basis of the evolution and host specialization in M. oryzae. [ABSTRACT FROM AUTHOR]

Summary: Interactions between Leptosphaeria maculans, causal agent of stem canker of oilseed rape, and its Brassica hosts are models of choice to explore the multiplicity of 'gene‐for‐gene' complementarities and how they diversified to increased complexity in the course of plant–pathogen co‐evolution. Here, we support this postulate by investigating the AvrLm10 avirulence that induces a resistance response when recognized by the Brassica nigra resistance gene Rlm10.Using genome‐assisted map‐based cloning, we identified and cloned two AvrLm10 candidates as two genes in opposite transcriptional orientation located in a subtelomeric repeat‐rich region of the genome. The AvrLm10 genes encode small secreted proteins and show expression profiles in planta similar to those of all L. maculans avirulence genes identified so far.Complementation and silencing assays indicated that both genes are necessary to trigger Rlm10 resistance. Three assays for protein–protein interactions showed that the two AvrLm10 proteins interact physically in vitro and in planta.Some avirulence genes are recognized by two distinct resistance genes and some avirulence genes hide the recognition specificities of another. Our L. maculans model illustrates an additional case where two genes located in opposite transcriptional orientation are necessary to induce resistance. Interestingly, orthologues exist for both L. maculans genes in other phytopathogenic species, with a similar genome organization, which may point to an important conserved effector function linked to heterodimerization of the two proteins. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii typically causes lifelong chronic infection and has been identified in a variety of intermediate and definitive hosts. Felids are capable of serving as both intermediate and definite hosts for T. gondii infection. However, there is no direct evidence to prove that servals are the intermediate host of T. gondii. In this study, T. gondii antibodies were detected in a serval by a modified agglutination test (titer, 1:200). Viable T. gondii was isolated from the striated muscles of the serval. This strain was further propagated in cell culture and designated as TgServalCHn1. Genetic characterization of DNA derived from cell culture was performed by RFLP‐PCR of 10 markers, as well as polymorphic ROP5 and ROP18 genes. Results showed that this strain of T. gondii belonged to the genotype ToxoDB#20. The ROP5 allele 4 and ROP18 allele 3 suggested that this strain was avirulent, which was further supported by infection in mice. Encephalitis, immune organ necrosis and focal mononuclear cell infiltration in multiple organs were the main pathology characteristics observed in BALB/C mice infected with the TgServalCHn1 strain. To our knowledge, the present study is the first to report the isolation of T. gondii from a serval, which gives direct evidence for servals serving as an intermediate host of T. gondii. The genotyping results revealed the presence of genotype ToxoDB#20 in central China, enriching the scope of the distribution of T. gondii genotypes in Asia. [ABSTRACT FROM AUTHOR]

Summary: The oomycete pathogens Phytophthora infestans and P. capsici cause significant crop losses world‐wide, threatening food security. In each case, pathogenicity factors, called RXLR effectors, contribute to virulence. Some RXLRs are perceived by resistance proteins to trigger host immunity, but our understanding of the demographic processes and adaptive evolution of pathogen virulence remains poor.Here, we describe PenSeq, a highly efficient enrichment sequencing approach for genes encoding pathogenicity determinants which, as shown for the infamous potato blight pathogen Phytophthora infestans, make up < 1% of the entire genome.PenSeq facilitates the characterization of allelic diversity in pathogen effectors, enabling evolutionary and population genomic analyses of Phytophthora species. Furthermore, PenSeq enables the massively parallel identification of presence/absence variations and sequence polymorphisms in key pathogen genes, which is a prerequisite for the efficient deployment of host resistance genes.PenSeq represents a cost‐effective alternative to whole‐genome sequencing and addresses crucial limitations of current plant pathogen population studies, which are often based on selectively neutral markers and consequently have limited utility in the analysis of adaptive evolution. The approach can be adapted to diverse microbes and pathogens. See also the Commentary on this article by Kale, 221: 1177–1179. [ABSTRACT FROM AUTHOR]

Oomycetes such as the potato blight pathogen Phytophthora infestans deliver RXLR effectors into plant cells to manipulate host processes and promote disease. Knowledge of where they localize inside host cells is important in understanding their function. Fifty-two P. infestans RXLR effectors (PiRXLRs) up-regulated during early stages of infection were expressed as fluorescent protein (FP) fusions inside cells of the model host Nicotiana benthamiana. FP–PiRXLR fusions were predominantly nucleo-cytoplasmic, nuclear, or plasma membrane-associated. Some also localized to the endoplasmic reticulum, mitochondria, peroxisomes, or microtubules, suggesting diverse sites of subcellular activity. Seven of the 25 PiRXLRs examined during infection accumulated at sites of haustorium penetration, probably due to co-localization with host target processes; Pi16663 (Avr1), for example, localized to Sec5-associated mobile bodies which showed perihaustorial accumulation. Forty-five FP–RXLR fusions enhanced pathogen leaf colonization when expressed in Nicotiana benthamiana, revealing that their presence was beneficial to infection. Co-expression of PiRXLRs that target and suppress different immune pathways resulted in an additive enhancement of colonization, indicating the potential to study effector combinations using transient expression assays. We provide a broad platform of high confidence P. infestans effector candidates from which to investigate the mechanisms, singly and in combination, by which this pathogen causes disease. [ABSTRACT FROM AUTHOR]

• Genome-wide analyses of the effector- and toxin-encoding genes were used to examine the phylogenetics and evolution of pathogenicity amongst diverse strains of Pseudomonas syringae causing bacterial canker of cherry (Prunus avium), including pathovars P. syringae pv morsprunorum (Psm) races 1 and 2, P. syringae pv syringae (Pss) and P. syringae pv avii. • Phylogenetic analyses revealed Psm races and P. syringae pv avii clades were distinct and were each monophyletic, whereas cherry-pathogenic strains of Pss were interspersed amongst strains from other host species. • A maximum likelihood approach was used to predict effectors associated with pathogenicity on cherry. Pss possesses a smaller repertoire of type III effectors but has more toxin biosynthesis clusters than Psm and P. syringae pv avii. Evolution of cherry pathogenicity was correlated with gain of genes such as hopAR1 and hopBB1 through putative phage transfer and horizontal transfer respectively. By contrast, loss of the avrPto/hopAB redundant effector group was observed in cherry-pathogenic clades. Ectopic expression of hopAB and hopC1 triggered the hypersensitive reaction in cherry leaves, confirming computational predictions. • Cherry canker provides a fascinating example of convergent evolution of pathogenicity that is explained by the mix of effector and toxin repertoires acting on a common host. [ABSTRACT FROM AUTHOR]

We investigated the involvement of two avirulence genes, avrA ( ripAA in unified nomenclature) and popP1 ( ripP1 in unified nomenclature), of Japanese Ralstonia solanacearum strains in the pathogenicity to tobacco. One virulent strain OE1-1 and four hypersensitive response (HR)-eliciting strains, 8107, MAFF 211471, MAFF 211496, and MAFF 301520, were used. While 8107 and MAFF 211471 contain popP1 , other three strains do not. When popP1 of strain 8107 was transferred into the virulent strain OE1-1, the transconjugant strain had significantly reduced virulence but did not become a HR-eliciting strain. We deleted avrA and/or popP1 from the HR-eliciting strains; all deletion mutants still elicited a HR and did not become virulent to tobacco, although leaf lesion appearance was delayed on infection with avrA mutants. These results indicate that although avrA and popP1 could be functional as avirulence determinants, other unidentified factors are necessary for full virulence or HR elicitation by Japanese R. solanacearum strains. [ABSTRACT FROM AUTHOR]

Summary: Leptosphaeria maculans, the causal agent of blackleg disease, interacts with Brassica napus (oilseed rape, canola) and other Brassica hosts in a gene‐for‐gene manner. The avirulence gene AvrLmJ1 has been cloned previously and shown to interact with an unidentified Brassica juncea resistance gene. In this study, we show that the AvrLmJ1 gene maps to the same position as the AvrLm5 locus. Furthermore, isolates complemented with the AvrLmJ1 locus confer avirulence towards B. juncea genotypes harbouring Rlm5. These findings demonstrate that AvrLmJ1 is AvrLm5 and highlight the need for shared resources to characterize accurately avirulence and/or resistance genes. [ABSTRACT FROM AUTHOR]

Summary: Recent advances in genomics and single‐cell analysis have demonstrated the extraordinary complexity reached by microbial populations within their hosts. Communities range from complex multispecies groups to homogeneous populations differentiating into lineages through genetic or non‐genetic mechanisms. Diversity within bacterial populations is recognized as a key driver of the evolution of animal pathogens. In plants, however, little is known about how interactions between different pathogenic and non‐pathogenic variants within the host impact on defence responses, or how the presence within a mixture may affect the development or the fate of each variant. Using confocal fluorescence microscopy, we analysed the colonization of the plant apoplast by individual virulence variants of Pseudomonas syringae within mixed populations. We found that non‐pathogenic variants can proliferate and even spread beyond the inoculated area to neighbouring tissues when in close proximity to pathogenic bacteria. The high bacterial concentrations reached at natural entry points promote such interactions during the infection process. We also found that a diversity of interactions take place at a cellular level between virulent and avirulent variants, ranging from dominant negative effects on proliferation of virulent bacteria to in trans suppression of defences triggered by avirulent bacteria. Our results illustrate the spatial dynamics and complexity of the interactions found within mixed infections, and their potential impact on pathogen evolution. [ABSTRACT FROM AUTHOR]

Summary: Phytopathogens translocate effector proteins into plant cells where they sabotage the host cellular machinery to promote infection. An individual pathogen can translocate numerous distinct effectors during the infection process to target an array of host macromolecules (proteins, metabolites, DNA, etc.) and manipulate them using a variety of enzymatic activities. In this review, we have surveyed the literature for effector targets and curated them to convey the range of functions carried out by phytopathogenic proteins inside host cells. In particular, we have curated the locations of effector targets, as well as their biological and molecular functions and compared these properties across diverse phytopathogens. This analysis validates previous observations about effector functions (e.g. immunosuppression), and also highlights some interesting features regarding effector specificity as well as functional diversification of phytopathogen virulence strategies. [ABSTRACT FROM AUTHOR]

Blackleg disease, caused by the ascomycete fungal pathogen Leptosphaeria maculans, is a devastating disease of canola (Brassica napus) in Australia, Canada and Europe. Although cultural strategies such as crop rotation, fungicide application, and tillage are adopted to control the disease, the most promising disease control strategy is the utilisation of resistant canola varieties. However, field populations of L. maculans display a high evolutionary potential and are able to overcome major resistance genes within a few years, making disease control relying on resistant varieties challenging. In the early 1990s, blackleg resistance gene Rlm3 was introduced into Canadian canola varieties and provided good resistance against the fungal populations until the early 2000s, when moderate to severe blackleg outbreaks were observed in some areas across western Canada. However, the breakdown of Rlm3 resistance was not reported until recently, based on studies on R genes present in Canadian canola varieties and the avirulence allele frequency in L. maculans populations in western Canada. The fact that Rlm3 was overcome by the evolution of fungal populations demands canola breeding programs in Canada to be prepared to develop canola varieties with diversified and efficient R genes. In addition, frequent monitoring of fungal populations can provide up-to-date guidance for proper resistance genes deployment. This literature review provides insights into the outbreaks and management of blackleg disease in Canada. [ABSTRACT FROM AUTHOR]

Bacterial phytopathogen type III secreted (T3S) effectors have been strongly implicated in altering the interaction of pathogens with host plants. Therefore, it is useful to characterize the whole effector repertoire of a pathogen to understand the interplay of effectors in plants. Pseudomonas syringae pv. actinidiae is a causal agent of kiwifruit canker disease. In this study, we generated an Agrobacterium-mediated transient expression library of YFP-tagged T3S effectors from two strains of Psa, Psa-NZ V13 and Psa-NZ LV5, in order to gain insight into their mode of action in Nicotiana tabacum and N. benthamiana. Determining the subcellular localization of effectors gives an indication of the possible host targets of effectors. A confocal microscopy assay detecting YFP-tagged Psa effectors revealed that the nucleus, cytoplasm and cell periphery are major targets of Psa effectors. Agrobacterium-mediated transient expression of multiple Psa effectors induced HR-like cell death (HCD) in Nicotiana spp., suggesting that multiple Psa effectors may be recognized by Nicotiana spp.. Virus-induced gene silencing (VIGS) of several known plant immune regulators, EDS1, NDR1, or SGT1 specified the requirement of SGT1 in HCD induced by several Psa effectors in N. benthamiana. In addition, the suppression activity of Psa effectors on HCD-inducing proteins and PTI was assessed. Psa effectors showed differential suppression activities on each HCD inducer or PTI. Taken together, our Psa effector repertoire analysis highlights the great diversity of T3S effector functions in planta.

In this paper we have characterized the lineage of two traits associated with the coat proteins (CPs) of the tombusvirids: Silencing suppression and HR elicitation in Nicotiana species. We considered that the tombusvirid CPs might collectively be considered an effector, with the CP of each CP-encoding species comprising a structural variant within the family. Thus, a phylogenetic analysis of the CP could provide insight into the evolution of a pathogen effector. The phylogeny of the CP of tombusvirids indicated that CP representatives of the family could be divided into four clades. In two separate clades the CP triggered a hypersensitive response (HR) in Nicotiana species of section Alatae but did not have silencing suppressor activity. In a third clade the CP had a silencing suppressor activity but did not have the capacity to trigger HR in Nicotiana species. In the fourth clade, the CP did not carry either function. Our analysis illustrates how structural changes that likely occurred in the CP effector of progenitors of the current genera led to either silencing suppressor activity, HR elicitation in select Nicotiana species, or neither trait.

Candidate effectors from lettuce downy mildew ( Bremia lactucae) enable high-throughput germplasm screening for the presence of resistance ( R) genes. The nonhost species Lactuca saligna comprises a source of B. lactucae R genes that has hardly been exploited in lettuce breeding. Its cross-compatibility with the host species L. sativa enables the study of inheritance of nonhost resistance ( NHR)., We performed transient expression of candidate RXLR effector genes from B. lactucae in a diverse Lactuca germplasm set. Responses to two candidate effectors ( BLR31 and BLN08) were genetically mapped and tested for co-segregation with disease resistance., BLN08 induced a hypersensitive response (HR) in 55% of the L. saligna accessions, but responsiveness did not co-segregate with resistance to Bl:24. BLR31 triggered an HR in 5% of the L. saligna accessions, and revealed a novel R gene providing complete B. lactucae race Bl:24 resistance. Resistant hybrid plants that were BLR31 nonresponsive indicated other unlinked R genes and/or nonhost QTLs., We have identified a candidate avirulence effector of B. lactucae ( BLR31) and its cognate R gene in L. saligna. Concurrently, our results suggest that R genes are not required for NHR of L. saligna. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa causes Pierce's disease (PD) on grapevines, leading to significant economic losses in grape and wine production. To further our understanding of X. fastidiosa virulence on grapevines, we examined the PD1311 gene, which encodes a putative acyl-coenzyme A (acyl-CoA) synthetase, and is highly conserved across Xylella species. It was determined that PD1311 is required for virulence, as the deletion mutant, ΔPD1311, was unable to cause disease on grapevines. The ΔPD1311 strain was impaired in behaviours known to be associated with PD development, including motility, aggregation and biofilm formation. ΔPD1311 also expressed enhanced sensitivity to H2O2 and polymyxin B, and showed reduced survival in grapevine sap, when compared with wild-type X. fastidiosa Temecula 1 (TM1). Following inoculation, ΔPD1311 could not be detected in grape shoots, which may be related to its altered growth and sensitivity phenotypes. Inoculation with ΔPD1311 2 weeks prior to TM1 prevented the development of PD in a significant fraction of vines and eliminated detectable levels of TM1. In contrast, vines inoculated simultaneously with TM1 and ΔPD1311 developed disease at the same level as TM1 alone. In these vines, TM1 populations were distributed similarly to populations in TM1-only inoculated plants. These findings suggest that, through an indirect mechanism, pretreatment of vines with ΔPD1311 suppresses pathogen population and disease. [ABSTRACT FROM AUTHOR]

Zymoseptoria tritici is the causal agent of Septoria tritici blotch, a major pathogen of wheat globally and the most damaging pathogen of wheat in Europe. A gene-for-gene ( GFG) interaction between Z. tritici and wheat cultivars carrying the Stb6 resistance gene has been postulated for many years, but the genes have not been identified., We identified AvrStb6 by combining quantitative trait locus mapping in a cross between two Swiss strains with a genome-wide association study using a natural population of c. 100 strains from France. We functionally validated AvrStb6 using ectopic transformations., AvrStb6 encodes a small, cysteine-rich, secreted protein that produces an avirulence phenotype on wheat cultivars carrying the Stb6 resistance gene. We found 16 nonsynonymous single nucleotide polymorphisms among the tested strains, indicating that AvrStb6 is evolving very rapidly. AvrStb6 is located in a highly polymorphic subtelomeric region and is surrounded by transposable elements, which may facilitate its rapid evolution to overcome Stb6 resistance., AvrStb6 is the first avirulence gene to be functionally validated in Z. tritici, contributing to our understanding of avirulence in apoplastic pathogens and the mechanisms underlying GFG interactions between Z. tritici and wheat. [ABSTRACT FROM AUTHOR]

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. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

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.

XopJ is a Xanthomonas type III effector protein that promotes bacterial virulence on susceptible pepper plants through the inhibition of the host cell proteasome and a resultant suppression of salicylic acid (SA) – dependent defense responses. We show here that Nicotiana benthamiana leaves transiently expressing XopJ display hypersensitive response (HR) –like symptoms when exogenously treated with SA. This apparent avirulence function of XopJ was further dependent on effector myristoylation as well as on an intact catalytic triad, suggesting a requirement of its enzymatic activity for HR-like symptom elicitation. The ability of XopJ to cause a HR-like symptom development upon SA treatment was lost upon silencing of SGT1 and NDR1, respectively, but was independent of EDS1 silencing, suggesting that XopJ is recognized by an R protein of the CC-NBS-LRR class. Furthermore, silencing of NPR1 abolished the elicitation of HR-like symptoms in XopJ expressing leaves after SA application. Measurement of the proteasome activity indicated that proteasome inhibition by XopJ was alleviated in the presence of SA, an effect that was not observed in NPR1 silenced plants. Our results suggest that XopJ - triggered HR-like symptoms are closely related to the virulence function of the effector and that XopJ follows a two-signal model in order to elicit a response in the non-host plant N. benthamiana.

The wheat stem rust fungus Puccinia graminis f. sp. tritici (Pgt), is one of the most destructive pathogens of wheat. In this study, a draft genome was built for a founder Australian Pgt isolate of pathotype (pt.) 21-0 (collected in 1954) by next generation DNA sequencing. A combination of reference-based assembly using the genome of the previously sequenced American Pgt isolate CDL 75-36-700-3 (p7a) and de novo assembly were performed resulting in a 92 Mbp reference genome for Pgt isolate 21-0. Approximately 13 Mbp of de novo assembled sequence in this genome is not present in the p7a reference assembly. This novel sequence is not specific to 21-0 as it is also present in three other Pgt rust isolates of independent origin.The new reference genome was subsequently used to build a pan-genome based on five Australian Pgt isolates. Transcriptomes from germinated urediniospores and haustoria were separately assembled for pt. 21-0 and comparison of gene expression profiles showed differential expression in ~10% of the genes each in germinated spores and haustoria. A total of 1,924 secreted proteins were predicted from the 21-0 transcriptome, of which 520 were classified as haustorial secreted proteins (HSPs). Comparison of 21-0 with two presumed clonal field derivatives of this lineage (collected in 1982 and 1984) that had evolved virulence on four additional resistance genes (Sr5, Sr11, Sr27, SrSatu) identified mutations in 25 HSP effector candidates, some of which could explain their novel virulence phenotypes.

Bacterial disease management is a challenge for modern agriculture due to rapid changes in pathogen populations. Genome sequences for hosts and pathogens provide detailed information that facilitates effectorbased breeding strategies. Tomato genotypes have gene-for-gene resistance to the bacterial spot pathogen Xanthomonas perforans. The bacterial spot populations in Florida shifted from tomato race 3 to 4, such that the corresponding tomato resistance gene no longer recognizes the effector protein AvrXv3. Genome sequencing showed variation in effector profiles among race 4 strains collected in 2006 and 2012 and compared with a race 3 strain collected in 1991. We examined variation in putative targets of resistance among Florida strains of X. perforans collected from 1991 to 2006. Consistent with race change, avrXv3 was present in race 3 strains but nonfunctional in race 4 strains due to multiple independent mutations. Effectors xopJ4 and avrBs2 were unchanged in all strains. The effector avrBsT was absent in race 3 strains collected in the 1990s but present in race 3 strains collected in 2006 and nearly all race 4 strains. These changes in effector profiles suggest that xopJ4 and avrBsT are currently the best targets for resistance breeding against bacterial spot in tomato. [ABSTRACT FROM AUTHOR]

'Gene-for-gene' theory predicts that gain of virulence by an avirulent pathogen on plants expressing resistance ( R) genes is associated with fitness loss in susceptible hosts. However, the validity of this prediction has been studied in only a few plant viral pathosystems. In this study, the Soybean mosaic virus (SMV)- Rsv4 pathosystem was exploited to test this prediction. In Rsv4-genotype soybeans, P3 of avirulent SMV strains provokes an as yet uncharacterized resistance mechanism that restricts the invading virus to the inoculated leaves. A single amino acid substitution in P3 functionally converts an avirulent to a virulent strain, suggesting that the genetic composition of P3 plays a crucial role in virulence on Rsv4-genotype soybeans. In this study, we examined the impact of gain of virulence mutation(s) on the fitness of virulent variants derived from three avirulent SMV strains in a soybean genotype lacking the Rsv4 gene. Our data demonstrate that gain of virulence mutation(s) by all avirulent viruses on Rsv4-genotype soybean is associated with a relative fitness loss in a susceptible host. The implications of this finding on the durable deployment of the Rsv4 gene in soybean are discussed. [ABSTRACT FROM AUTHOR]

Extending the durability of plant resistance genes towards fungal pathogens is a major challenge. We identified and investigated the relationship between two avirulence genes of Leptosphaeria maculans, AvrLm3 and AvrLm4-7. When an isolate possesses both genes, the Rlm3-mediated resistance of oilseed rape ( Brassica napus) is not expressed due to the presence of AvrLm4-7 but virulent isolates toward Rlm7 recover the AvrLm3 phenotype., Combining genetic and genomic approaches (genetic mapping, RNA-seq, BAC (bacterial artificial chromosome) clone sequencing and de novo assembly) we cloned AvrLm3, a telomeric avirulence gene of L. maculans. AvrLm3 is located in a gap of the L. maculans reference genome assembly, is surrounded by repeated elements, encodes for a small secreted cysteine-rich protein and is highly expressed at early infection stages., Complementation and silencing assays validated the masking effect of AvrLm4-7 on AvrLm3 recognition by Rlm3 and we showed that the presence of AvrLm4-7 does not impede AvrLm3 expression in planta. Y2H assays suggest the absence of physical interaction between the two avirulence proteins., This unusual interaction is the basis for field experiments aiming to evaluate strategies that increase Rlm7 durability. [ABSTRACT FROM AUTHOR]

Blackleg disease caused by the fungus Leptosphaeria maculans is the most important disease of canola worldwide. The impact of this disease on the development of the Australian canola industry, particularly over the last 20 years, is discussed. Deployment of a range of disease control measures has resulted in a thriving canola industry with production now approaching 4 million tonnes annually. Discoveries about disease mechanisms and key plant and fungal genes are described. Analysis of the L. maculans genome sequence has enabled an understanding of how fungal populations can evolve rapidly to overcome disease resistance bred into canola cultivars. [ABSTRACT FROM AUTHOR]

The recent resurgence of wheat stem rust caused by new virulent races of Puccinia graminis f. sp. tritici (Pgt) poses a threat to food security. These concerns have catalyzed an extensive global effort toward controlling this disease. Substantial research and breeding programs target the identification and introduction of new stem rust resistance (Sr) genes in cultivars for genetic protection against the disease. Such resistance genes typically encode immune receptor proteins that recognize specific components of the pathogen, known as avirulence (Avr) proteins. A significant drawback to deploying cultivars with single Sr genes is that they are often overcome by evolution of the pathogen to escape recognition through alterations in Avr genes. Thus, a key element in achieving durable rust control is the deployment of multiple effective Sr genes in combination, either through conventional breeding or transgenic approaches, to minimize the risk of resistance breakdown. In this situation, evolution of pathogen virulence would require changes in multiple Avr genes in order to bypass recognition. However, choosing the optimal Sr gene combinations to deploy is a challenge that requires detailed knowledge of the pathogen Avr genes with which they interact and the virulence phenotypes of Pgt existing in nature. Identifying specific Avr genes from Pgt will provide screening tools to enhance pathogen virulence monitoring, assess heterozygosity and propensity for mutation in pathogen populations, and confirm individual Sr gene functions in crop varieties carrying multiple effective resistance genes. Toward this goal, much progress has been made in assembling a high quality reference genome sequence for Pgt, as well as a Pan-genome encompassing variation between multiple field isolates with diverse virulence spectra. In turn this has allowed prediction of Pgt effector gene candidates based on known features of Avr genes in other plant pathogens, including the related flax rust fungus. Upregulation of gene expression in haustoria and evidence for diversifying selection are two useful parameters to identify candidate Avr genes. Recently, we have also applied machine learning approaches to agnostically predict candidate effectors. Here, we review progress in stem rust pathogenomics and approaches currently underway to identify Avr genes recognized by wheat Sr genes. [ABSTRACT FROM AUTHOR]

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.

• Avirulent Zymoseptoria tritici strains can reproduce sexually in wheat plants carrying the corresponding resistant gene (R). • Symptomatic infection of plant tissues is not essential for a strain to reproduce sexually. • Avirulent strains can be maintained asymptomatically on or in leaf tissues of R plants for long enough to reproduce sexually. • Crosses of virulent strains with virulent and avirulent strains in R plants can produce a similar sized offspring population. • Several possible scenarios for sexual crosses can explain the maintenance of avirulence in Zymoseptoria tritici populations. Little is known about the impact of host immunity on sexual reproduction in fungal pathogens. In particular, it is unclear whether crossing requires both sexual partners to infect living plant tissues. We addressed this issue in a three-year experiment investigating different scenarios of Zymoseptoria tritici crosses according to the virulence ('vir') or avirulence ('avr') of the parents against a qualitative resistance gene. Co-inoculations ('vir × vir', 'avr × vir', 'avr × avr') and single inoculations were performed on a wheat cultivar carrying the Stb16q resistance gene (Cellule) and a susceptible cultivar (Apache), in the greenhouse. We assessed the intensity of asexual reproduction by scoring disease severity, and the intensity of sexual reproduction by counting the ascospores discharged from wheat residues. As expected, disease severity was more intense on Cellule for 'vir × vir' co-inoculations than for 'avr × vir' co-inoculations, with no disease for 'avr × avr'. However, all types of co-inoculation yielded sexual offspring, whether or not the parental strains caused plant symptoms. Parenthood was confirmed by genotyping (SSR markers), and the occurrence of crosses between (co-)inoculated and exogenous strains (other strains from the experiment, or from far away) was determined. We showed that symptomatic asexual infection was not required for a strain to participate in sexual reproduction, and deduced from this result that avirulent strains could be maintained asymptomatically "on" or "in" leaf tissues of plants carrying the corresponding resistant gene for long enough to reproduce sexually. In two of the three years, the intensity of sexual reproduction did not differ between the three types of co-inoculation in Cellule, suggesting that crosses involving avirulent strains are not anecdotal. We discuss the possible mechanisms explaining the maintenance of avirulence in Z. tritici populations and the potential impact of particular resistance deployments such as cultivar mixtures for limiting resistance breakdown. [ABSTRACT FROM AUTHOR]

Plant pathogens cause severe losses to crop plants and threaten global food production. One striking example is the wheat stem rust fungus, Puccinia graminis f. sp. tritici, which can rapidly evolve new virulent pathotypes in response to resistant host lines. Like several other filamentous fungal and oomycete plant pathogens, its genome features expanded gene families that have been implicated in host-pathogen interactions, possibly encoding effector proteins that interact directly with target host defence proteins. Previous efforts to understand virulence largely relied on the prediction of secreted, small and cysteine-rich proteins as candidate effectors and thus delivered an overwhelming number of candidates. Here, we implement an alternative analysis strategy that uses the signal of adaptive evolution as a line of evidence for effector function, combined with comparative information and expression data. We demonstrate that in planta up-regulated genes that are rapidly evolving are found almost exclusively in pathogen-associated gene families, affirming the impact of host-pathogen co-evolution on genome structure and the adaptive diversification of specialised gene families. In particular, we predict 42 effector candidates that are conserved only across pathogens, induced during infection and rapidly evolving. One of our top candidates has recently been shown to induce genotype-specific hypersensitive cell death in wheat. This shows that comparative genomics incorporating the evolutionary signal of adaptation is powerful for predicting effector candidates for laboratory verification. Our system can be applied to a wide range of pathogens and will give insight into host-pathogen dynamics, ultimately leading to progress in strategies for disease control.

To establish a successful infection plant viruses have to overcome a defence system composed of several layers. This review will overview the various strategies plants employ to combat viral infections with main emphasis on the current status of single dominant resistance (R) genes identified against plant viruses and the corresponding avirulence (Avr) genes identified so far. The most common models to explain the mode of action of dominant R genes will be presented. Finally, in brief the hypersensitive response (HR) and extreme resistance (ER), and the functional and structural similarity of R-genes to sensors of innate immunity in mammalian cell systems will be described.

Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimise parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analysed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their hosts.

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.

Plant-invading microbes betray their presence to a plant by exposure of antigenic molecules such as small, secreted proteins called 'effectors'. In Fusarium oxysporum f. sp . lycopersici ( Fol) we identified a pair of effector gene candidates, AVR2- SIX5, whose expression is controlled by a shared promoter., The pathogenicity of AVR2 and SIX5 Fol knockouts was assessed on susceptible and resistant tomato ( Solanum lycopersicum) plants carrying I-2. The I-2 NB- LRR protein confers resistance to Fol races carrying AVR2., Like Avr2, Six5 was found to be required for full virulence on susceptible plants. Unexpectedly, each knockout could breach I-2-mediated disease resistance. So whereas Avr2 is sufficient to induce I-2-mediated cell death, Avr2 and Six5 are both required for resistance. Avr2 and Six5 interact in yeast two-hybrid assays as well as in planta. Six5 and Avr2 accumulate in xylem sap of plants infected with the reciprocal knockouts, showing that lack of I-2 activation is not due to a lack of Avr2 accumulation in the SIX5 mutant., The effector repertoire of a pathogen determines its host specificity and its ability to manipulate plant immunity. Our findings challenge an oversimplified interpretation of the gene-for-gene model by showing requirement of two fungal genes for immunity conferred by one resistance gene. [ABSTRACT FROM AUTHOR]