ZymoSoups: A high-throughput forward genetics method for rapid identification of virulence genes in Zymoseptoria tritici.

Septoria tritici blotch is caused by the fungus Zymoseptoria tritici and poses a major threat to wheat productivity. There are over twenty mapped loci in wheat that confer strong (gene-for-gene) resistance against this pathogen, however the corresponding genes in Z. tritici that confer virulence against distinct R genes remain largely unknown. In this study, we developed a rapid forward genetics methodology to identify genes that enable Z. tritici to gain virulence on previously resistant wheat varieties. We used the known gene-for-gene interaction between Stb6 and AvrStb6 as a proof-of-concept that this method could quickly recover single candidate virulence genes. We subjected the avirulent Z. tritici strain IPO323, which carries the recognized AvrStb6 allele, to UV mutagenesis and generated a library of over 66,000 mutants. We screened these mutants on leaves of the resistant wheat variety Cadenza, in mixtures (soups) ranging from 100-500 mutants per soup. We identified five soups with a gain-of-virulence (GoV) phenotype relative to the IPO323 parental strain and re-sequenced 18 individual isolates, including four control isolates and two mutants lacking virulence, when screened individually. Of the 12 confirmed GoV mutants, one had a single nucleotide polymorphism (SNP) in the AvrStb6 coding region. The other 11 GoV mutants exhibited large (~70Kb) deletions at the end of chromosome 5, including the AvrStb6 locus. Our findings demonstrate the efficiency of this forward genetic approach in elucidating the genetic basis of qualitative resistance to Z. tritici and the potential to rapidly identify other, currently unknown, Avr genes in this pathogen.

The Egyptian wheat cultivar Gemmeiza-12 is a source of resistance against the fungus Zymoseptoria tritici.

BACKGROUND: Wheat is one of the world's most important cereal crops. However, the fungal pathogen Zymoseptoria tritici can cause disease epidemics, leading to reduced yields. With climate change and development of new agricultural areas with suitable environments, Z. tritici may advance into geographical areas previously unaffected by this pathogen. It is currently unknown how Egyptian wheat will perform in the face of this incoming threat. This project aimed to assess the resistance of Egyptian wheat germplasm to Z. tritici, to identify cultivars with high levels of resistance and characterise the mechanism(s) of resistance present in these cultivars. RESULTS: Eighteen Egyptian wheat cultivars were screened against two Z. tritici model isolates and exhibited a wide spectrum of responses. This ranged from resistance to complete susceptibility to one or both isolates tested. The most highly resistant cultivars from the initial screen were then tested under two environmental conditions against modern UK field isolates. Disease levels under UK-like conditions were higher, however, symptom development on the cultivar Gemmeiza-12 was noticeably slower than on other Egyptian wheats. The robustness of the resistance shown by Gemmeiza-12 was confirmed in experiments mimicking Egyptian environmental conditions, where degree of Z. tritici infection was lower. The Kompetitive allele-specific PCR (KASP) diagnostic assay suggested the presence of an Stb6 resistant allele in several Egyptian wheats including Gemmeiza-12. Infection assays using the IPO323 WT and IPO323ΔAvrStb6 mutant confirmed the presence of Stb6 in several Egyptian cultivars including Gemmeiza-12. Confocal fluorescence microscopy demonstrated that growth of the IPO323 strain is blocked at the point of stomatal penetration on Gemmeiza-12, consistent with previous reports of Stb gene mediated resistance. In addition to this R-gene mediated resistance, IPO323 spores showed lower adherence to leaves of Gemmeiza-12 compared to UK wheat varieties, suggesting other aspects of leaf physiology may also contribute to the resistance phenotype of this cultivar. CONCLUSION: These results indicate that Gemmeiza-12 will be useful in future breeding programs where improved resistance to Z. tritici is a priority.

Characterization of an antimicrobial and phytotoxic ribonuclease secreted by the fungal wheat pathogen Zymoseptoria tritici.

The fungus Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB) disease of wheat leaves. Zymoseptoria tritici secretes many functionally uncharacterized effector proteins during infection. Here, we characterized a secreted ribonuclease (Zt6) with an unusual biphasic expression pattern. Transient expression systems were used to characterize Zt6, and mutants thereof, in both host and non-host plants. Cell-free protein expression systems monitored the impact of Zt6 protein on functional ribosomes, and in vitro assays of cells treated with recombinant Zt6 determined toxicity against bacteria, yeasts and filamentous fungi. We demonstrated that Zt6 is a functional ribonuclease and that phytotoxicity is dependent on both the presence of a 22-amino-acid N-terminal 'loop' region and its catalytic activity. Zt6 selectively cleaves both plant and animal rRNA species, and is toxic to wheat, tobacco, bacterial and yeast cells, but not to Z. tritici itself. Zt6 is the first Z. tritici effector demonstrated to have a likely dual functionality. The expression pattern of Zt6 and potent toxicity towards microorganisms suggest that, although it may contribute to the execution of wheat cell death, it is also likely to have an important secondary function in antimicrobial competition and niche protection.

Apoplastic recognition of multiple candidate effectors from the wheat pathogen Zymoseptoria tritici in the nonhost plant Nicotiana benthamiana.

The fungus Zymoseptoria tritici is a strictly apoplastic, host-specific pathogen of wheat leaves and causal agent of septoria tritici blotch (STB) disease. All other plants are considered nonhosts, but the mechanism of nonhost resistance (NHR) to Z. tritici has not been addressed previously. We sought to develop Nicotiana benthamiana as a system to study NHR against Z. tritici. Fluorescence microscopy and quantitative reverse transcription polymerase chain reactions were used to establish the interaction between Z. tritici and N. benthamiana. Agrobacterium-mediated transient expression was used to screen putative Z. tritici effector genes for recognition in N. benthamiana, and virus-induced gene silencing (VIGS) was employed to determine the role of two receptor-like kinases (RLKs), NbBAK1 and NbSOBIR1, in Z. tritici effector recognition. Numerous Z. tritici putative effectors (14 of 63 tested) induced cell death or chlorosis in N. benthamiana. For most, phenotypes were light-dependent and required effector secretion to the leaf apoplastic space. Moreover, effector-induced host cell death was dependent on NbBAK1 and NbSOBIR1. Our results indicate widespread recognition of apoplastic effectors from a wheat-infecting fungal pathogen in a taxonomically distant nonhost plant species presumably by cell surface immune receptors. This suggests that apoplastic recognition of multiple nonadapted pathogen effectors may contribute to NHR.

An array of Zymoseptoria tritici effectors suppress plant immune responses.

Zymoseptoria tritici is the most economically significant fungal pathogen of wheat in Europe. However, despite the importance of this pathogen, the molecular interactions between pathogen and host during infection are not well understood. Herein, we describe the use of two libraries of cloned Z. tritici effectors that were screened to identify effector candidates with putative pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI)-suppressing activity. The effectors from each library were transiently expressed in Nicotiana benthamiana, and expressing leaves were treated with bacterial or fungal PAMPs to assess the effectors' ability to suppress reactive oxygen species (ROS) production. From these screens, numerous effectors were identified with PTI-suppressing activity. In addition, some effectors were able to suppress cell death responses induced by other Z. tritici secreted proteins. We used structural prediction tools to predict the putative structures of all of the Z. tritici effectors and used these predictions to examine whether there was enrichment of specific structural signatures among the PTI-suppressing effectors. From among the libraries, multiple members of the killer protein-like 4 (KP4) and killer protein-like 6 (KP6) effector families were identified as PTI suppressors. This observation is intriguing, as these protein families were previously associated with antimicrobial activity rather than virulence or host manipulation. This data provides mechanistic insight into immune suppression by Z. tritici during infection and suggests that, similar to biotrophic pathogens, this fungus relies on a battery of secreted effectors to suppress host immunity during early phases of colonization.

Resistance of Arabidopsis thaliana to the green peach aphid, Myzus persicae, involves camalexin and is regulated by microRNAs.

· Small RNAs play important roles in resistance to plant viruses and the complex responses against pathogens and leaf-chewing insects. · We investigated whether small RNA pathways are involved in Arabidopsis resistance against a phloem-feeding insect, the green peach aphid (Myzus persicae). We used a 2-wk fecundity assay to assess aphid performance on Arabidopsis RNA silencing and defence pathway mutants. Quantitative real-time polymerase chain reaction was used to monitor the transcriptional activity of defence-related genes in plants of varying aphid susceptibility. High-performance liquid chromatography-mass spectrometry was employed to measure the accumulation of the antimicrobial compound camalexin. Artificial diet assays allowed the assessment of the effect of camalexin on aphid performance. · Myzus persicae produces significantly less progeny on Arabidopsis microRNA (miRNA) pathway mutants. Plants unable to process miRNAs respond to aphid infestation with increased induction of PHYTOALEXIN DEFICIENT3 (PAD3) and production of camalexin. Aphids ingest camalexin when feeding on Arabidopsis and are more successful on pad3 and cyp79b2/cyp79b3 mutants defective in camalexin production. Aphids produce less progeny on artificial diets containing camalexin. · Our data indicate that camalexin functions beyond antimicrobial defence to also include hemipteran insects. This work also highlights the extensive role of the miRNA-mediated regulation of secondary metabolic defence pathways with relevance to resistance against a hemipteran pest.

Induction of distinct plant cell death programs by secreted proteins from the wheat pathogen Zymoseptoria tritici.

Cell death processes in eukaryotes shape normal development and responses to the environment. For plant-microbe interactions, initiation of host cell death plays an important role in determining disease outcomes. Cell death pathways are frequently initiated following detection of pathogen-derived molecules which can lead to resistance or susceptibility to disease depending on pathogen lifestyle. We previously identified several small secreted proteins (SSPs) from the wheat-infecting fungus Zymoseptoria tritici that induce rapid cell death in Nicotiana benthamiana following Agrobacterium-mediated delivery and expression (agroinfiltration). Here we investigated whether the execution of host cells was mechanistically similar in response to different Z. tritici SSPs. Using RNA sequencing, we found that transient expression of four Z. tritici SSPs led to massive transcriptional reprogramming within 48 h of agroinfiltration. We observed that distinct host gene expression profiles were induced dependent on whether cell death occurs in a cell surface immune receptor-dependent or -independent manner. These gene expression profiles involved differential transcriptional networks mediated by WRKY, NAC and MYB transcription factors. In addition, differential expression of genes belonging to different classes of receptor-like proteins and receptor-like kinases was observed. These data suggest that different Z. tritici SSPs trigger differential transcriptional reprogramming in plant cells.

Scaling-up to understand tree-pathogen interactions: A steep, tough climb or a walk in the park?

Plants have proficient tools that allow them to survive interactions with pathogens. Upon attack, they respond with specific countermeasures, which are controlled by the immune system. However, defences can fail and this failure exposes plants to fast-spreading devastation. Trees face similar challenges to other plants and their immune system allows them to mount defences against pathogens. However, their slow growth, longevity, woodiness, and size can make trees a challenging system to study. Here, we review scientific successes in plant systems, highlight the key challenges and describe the enormous opportunities for pathology research in trees. We discuss the benefits that scaling-up our understanding on tree-pathogen interactions can provide in the fight against plant pathogenic threats.

Food security in 2044: How do we control the fungal threat?

Plant fungal pathogens place considerable strain on agricultural productivity and threaten global food security. In recent decades, advances in crop breeding, farming practice and the agrochemical industry have allowed crop yields to keep pace with food demand. In this opinion article, we speculate on which recent technological advances will allow us to maintain this situation into the future. We take inspiration that it is 25 y since the first plant disease resistance genes were cloned, and imagine if and how agricultural control of pathogens will be achieved by the year 2044. We examine which technologies are best poised to make the jump from lab bench to field application, and propose that future control measures will likely depend on effective integrated disease management.

sRNA Profiling Combined With Gene Function Analysis Reveals a Lack of Evidence for Cross-Kingdom RNAi in the Wheat - Zymoseptoria tritici Pathosystem.

Cross-kingdom small RNA (sRNA) silencing has recently emerged as a mechanism facilitating fungal colonization and disease development. Here we characterized RNAi pathways in Zymoseptoria tritici, a major fungal pathogen of wheat, and assessed their contribution to pathogenesis. Computational analysis of fungal sRNA and host mRNA sequencing datasets was used to define the global sRNA populations in Z. tritici and predict their mRNA targets in wheat. 389 in planta-induced sRNA loci were identified. sRNAs generated from some of these loci were predicted to target wheat mRNAs including those potentially involved in pathogen defense. However, molecular approaches failed to validate targeting of selected wheat mRNAs by fungal sRNAs. Mutant strains of Z. tritici carrying deletions of genes encoding key components of RNAi such as Dicer-like (DCL) and Argonaute (AGO) proteins were generated, and virulence bioassays suggested that these are dispensable for full infection of wheat. Nonetheless, our results did suggest the existence of non-canonical DCL-independent pathway(s) for sRNA biogenesis in Z. tritici. dsRNA targeting essential fungal genes applied in vitro or generated from an RNA virus vector in planta in a procedure known as HIGS (Host-Induced Gene Silencing) was ineffective in preventing Z. tritici growth or disease. We also demonstrated that Z. tritici is incapable of dsRNA uptake. Collectively, our data suggest that RNAi approaches for gene function analyses in this fungal species and potentially also as a control measure may not be as effective as has been demonstrated for some other plant pathogenic fungi.

Rapid transcriptional plasticity of duplicated gene clusters enables a clonally reproducing aphid to colonise diverse plant species.

BACKGROUND: The prevailing paradigm of host-parasite evolution is that arms races lead to increasing specialisation via genetic adaptation. Insect herbivores are no exception and the majority have evolved to colonise a small number of closely related host species. Remarkably, the green peach aphid, Myzus persicae, colonises plant species across 40 families and single M. persicae clonal lineages can colonise distantly related plants. This remarkable ability makes M. persicae a highly destructive pest of many important crop species. RESULTS: To investigate the exceptional phenotypic plasticity of M. persicae, we sequenced the M. persicae genome and assessed how one clonal lineage responds to host plant species of different families. We show that genetically identical individuals are able to colonise distantly related host species through the differential regulation of genes belonging to aphid-expanded gene families. Multigene clusters collectively upregulate in single aphids within two days upon host switch. Furthermore, we demonstrate the functional significance of this rapid transcriptional change using RNA interference (RNAi)-mediated knock-down of genes belonging to the cathepsin B gene family. Knock-down of cathepsin B genes reduced aphid fitness, but only on the host that induced upregulation of these genes. CONCLUSIONS: Previous research has focused on the role of genetic adaptation of parasites to their hosts. Here we show that the generalist aphid pest M. persicae is able to colonise diverse host plant species in the absence of genetic specialisation. This is achieved through rapid transcriptional plasticity of genes that have duplicated during aphid evolution.

The Potato Aphid Salivary Effector Me47 Is a Glutathione-S-Transferase Involved in Modifying Plant Responses to Aphid Infestation.

Polyphagous aphid pests cause considerable economic damage to crop plants, primarily through the depletion of photoassimilates and transfer of viruses. The potato aphid (Macrosiphum euphorbiae) is a notable pest of solanaceous crops, however, the molecular mechanisms that underpin the ability to colonize these hosts are unknown. It has recently been demonstrated that like other aphid species, M. euphorbiae injects a battery of salivary proteins into host plants during feeding. It is speculated that these proteins function in a manner analagous to secreted effectors from phytopathogenic bacteria, fungi and oomycetes. Here, we describe a novel aphid effector (Me47) which was identified from the potato aphid salivary secretome as a putative glutathione-S-transferase (GST). Expression of Me47 in Nicotiana benthamiana enhanced reproductive performance of green peach aphid (Myzus persicae). Similarly, delivery of Me47 into leaves of tomato (Solanum lycopersicum) by Pseudomonas spp. enhanced potato aphid fecundity. In contrast, delivery of Me47 into Arabidopsis thaliana reduced GPA reproductive performance, indicating that Me47 impacts the outcome of plant-aphid interactions differently depending on the host species. Delivery of Me47 by the non-pathogenic Pseudomonas fluorescens revealed that Me47 protein or activity triggers defense gene transcriptional upregulation in tomato but not Arabidopsis. Recombinant Me47 was purified and demonstrated to have GST activity against two specific isothiocyanates (ITCs), compounds implicated in herbivore defense. Whilst GSTs have previously been associated with development of aphid resistance to synthetic insecticides, the findings described here highlight a novel function as both an elicitor and suppressor of plant defense when delivered into host tissues.

Dissecting the Molecular Interactions between Wheat and the Fungal Pathogen Zymoseptoria tritici.

The Dothideomycete fungus Zymoseptoria tritici (previously known as Mycosphaerella graminicola and Septoria tritici) is the causative agent of Septoria tritici leaf blotch (STB) disease of wheat (Triticum aestivum L.). In Europe, STB is the most economically damaging disease of wheat, with an estimated ∼€1 billion per year in fungicide expenditure directed toward its control. Here, an overview of our current understanding of the molecular events that occur during Z. tritici infection of wheat leaves is presented. On the host side, this includes the contribution of (1) the pathogen-associated molecular pattern-triggered immunity (PTI) layer of plant defense, and (2) major Stb loci for resistance against Z. tritici. On the pathogen side of the interaction, we consolidate evidence from recent bioinformatic, transcriptomic and proteomic studies that begin to explain the contribution of Z. tritici effector proteins to the biphasic lifestyle of the fungus. This includes the discovery of chitin-binding proteins in the Z. tritici secretome, which contribute to evasion of immune surveillance by this pathogen, and the possible existence of 'necrotrophic' effectors from Z. tritici, which may actively stimulate host recognition in a manner similar to related necrotrophic fungal pathogens. We finish by speculating on how some of these recent fundamental discoveries might be harnessed to help improve resistance to STB in the world's second largest food crop.