Jason J. Rudd

Finished Genome of the Fungal Wheat Pathogen Mycosphaerella graminicola Reveals Dispensome Structure, Chromosome Plasticity, and Stealth Pathogenesis

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed “mesosynteny” is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.

Pep-13, a plant defense-inducing pathogen-associated pattern from Phytophthora transglutaminases.

Innate immunity, an ancient form of defense against microbial infection, is well described for animals and is also suggested to be important for plants. Discrimination from self is achieved through receptors that recognize pathogen‐associated molecular patterns (PAMPs) not found in the host. PAMPs are evolutionarily conserved structures which are functionally important and, thus, not subject to frequent mutation. Here we report that the previously described peptide elicitor of defense responses in parsley, Pep‐13, constitutes a surface‐exposed fragment within a novel calcium‐dependent cell wall transglutaminase (TGase) from Phytophthora sojae. TGase transcripts and TGase activity are detectable in all Phytophthora species analyzed, among which are some of the most destructive plant pathogens. Mutational analysis within Pep‐13 identified the same amino acids indispensable for both TGase and defense‐eliciting activity. Pep‐13, conserved among Phytophthora TGases, activates defense in parsley and potato, suggesting its function as a genus‐specific recognition determinant for the activation of plant defense in host and non‐host plants. In summary, plants may recognize PAMPs with characteristics resembling those known to trigger innate immune responses in animals.

Analysis of Two in Planta Expressed LysM Effector Homologs from the Fungus Mycosphaerella graminicola Reveals Novel Functional Properties and Varying Contributions to Virulence on Wheat

Secreted effector proteins enable plant pathogenic fungi to manipulate host defenses for successful infection. Mycosphaerella graminicola causes Septoria tritici blotch disease of wheat (Triticum aestivum) leaves. Leaf infection involves a long (approximately 7 d) period of symptomless intercellular colonization prior to the appearance of necrotic disease lesions. Therefore, M. graminicola is considered as a hemibiotrophic (or necrotrophic) pathogen. Here, we describe the molecular and functional characterization of M. graminicola homologs of Ecp6 (for extracellular protein 6), the Lysin (LysM) domain-containing effector from the biotrophic tomato (Solanum lycopersicum) leaf mold fungus Cladosporium fulvum, which interferes with chitin-triggered immunity in plants. Three LysM effector homologs are present in the M. graminicola genome, referred to as Mg3LysM, Mg1LysM, and MgxLysM. Mg3LysM and Mg1LysM genes were strongly transcriptionally up-regulated specifically during symptomless leaf infection. Both proteins bind chitin; however, only Mg3LysM blocked the elicitation of chitin-induced plant defenses. In contrast to C. fulvum Ecp6, both Mg1LysM and Mg3LysM also protected fungal hyphae against plant-derived hydrolytic enzymes, and both genes show significantly more nucleotide polymorphism giving rise to nonsynonymous amino acid changes. While Mg1LysM deletion mutant strains of M. graminicola were fully pathogenic toward wheat leaves, Mg3LysM mutant strains were severely impaired in leaf colonization, did not trigger lesion formation, and were unable to undergo asexual sporulation. This virulence defect correlated with more rapid and pronounced expression of wheat defense genes during the symptomless phase of leaf colonization. These data highlight different functions for MgLysM effector homologs during plant infection, including novel activities that distinguish these proteins from C. fulvum Ecp6.

Transcriptional Adaptation of Mycosphaerella graminicola to Programmed Cell Death (PCD) of Its Susceptible Wheat Host

Many important fungal pathogens of plants spend long periods (days to weeks) of their infection cycle in symptomless association with living host tissue, followed by a sudden transition to necrotrophic feeding as host tissue death occurs. Little is known about either the host responses associated with this sudden transition or the specific adaptations made by the pathogen to invoke or tolerate it. We are studying a major host-specific fungal pathogen of cultivated wheat, Septoria tritici (teleomorph Mycosphaerella graminicola). Here, we describe the host responses of wheat leaves infected with M. graminicola during the development of disease symptoms and use microarray transcription profiling to identify adaptive responses of the fungus to its changing environment. We show that symptom development on a susceptible host genotype has features reminiscent of the hypersensitive response, a rapid and strictly localized form of host programmed cell death (PCD) more commonly associated with disease-resistance mechanisms. The initiation and advancement of this host response is associated with a loss of cell-membrane integrity and dramatic increases in apoplastic metabolites and the rate of fungal growth. Microarray analysis of the fungal genes differentially expressed before and after the onset of host PCD supports a transition to more rapid growth. Specific physiological adaptation of the fungus is also revealed with respect to membrane transport, chemical and oxidative stress mechanisms, and metabolism. Our data support the hypothesis that host plant PCD plays an important role in susceptibility towards fungal pathogens with necrotrophic lifestyles.

Dynamic Changes in the Localization of MAPK Cascade Components Controlling Pathogenesis-related (PR) Gene Expression during Innate Immunity in Parsley

The activation of mitogen-activated protein kinase (MAPK) cascades is an important mechanism for stress adaptation through the control of gene expression in mammals, yeast, and plants. MAPK activation has emerged as a common mechanism by which plants trigger pathogen defense responses following innate immune recognition of potential microbial pathogens. We are studying the non-host plant defense response of parsley to attempted infection by Phytophthora species using an experimental system of cultured parsley cells and the Phytophthora-derived Pep-13 peptide elicitor. Following receptor-mediated recognition of this peptide, parsley cells trigger a multifaceted innate immune response, involving the activation of three MAPKs that have been shown to function in the oxidative burst-independent activation of defense gene expression. Using this same experimental model we now report the identification of a MAPK kinase (MAPKK) that functions upstream in this pathway. This kinase, referred to as PcMKK5 based on sequence similarity to Arabidopsis thaliana AtMKK5, is activated in parsley cells following Pep-13 treatment and functions as an in vivo activator of all three MAPKs previously shown to be involved in this response. Gain- and loss-of-function mutant versions of PcMKK5, when used in protoplast co-transfection assays, demonstrated that kinase activity of PcMKK5 is required for PR gene promoter activation following Pep-13 treatment. Furthermore, using specific antibodies and immunofluorescent labeling, we demonstrate that activation of MAPKs in parsley cells correlates with an increase in their nuclear localization, which is not detectable for activated PcMKK5. These results suggest that activation of gene expression through MAPK cascades during innate immune responses in plants involves dynamic changes in the localization of the proteins involved, which may reflect the distribution of key protein substrates for the activated MAPKs.

Transcriptome and Metabolite Profiling of the Infection Cycle of Zymoseptoria tritici on Wheat Reveals a Biphasic Interaction with Plant Immunity Involving Differential Pathogen Chromosomal Contributions and a Variation on the Hemibiotrophic Lifestyle Definition

The temporal dynamics of Zymoseptoria tritici reproduction on Triticum aestivum involves a biphasic manipulation of plant defense responses. The hemibiotrophic fungus Zymoseptoria tritici causes Septoria tritici blotch disease of wheat (Triticum aestivum). Pathogen reproduction on wheat occurs without cell penetration, suggesting that dynamic and intimate intercellular communication occurs between fungus and plant throughout the disease cycle. We used deep RNA sequencing and metabolomics to investigate the physiology of plant and pathogen throughout an asexual reproductive cycle of Z. tritici on wheat leaves. Over 3,000 pathogen genes, more than 7,000 wheat genes, and more than 300 metabolites were differentially regulated. Intriguingly, individual fungal chromosomes contributed unequally to the overall gene expression changes. Early transcriptional down-regulation of putative host defense genes was detected in inoculated leaves. There was little evidence for fungal nutrient acquisition from the plant throughout symptomless colonization by Z. tritici, which may instead be utilizing lipid and fatty acid stores for growth. However, the fungus then subsequently manipulated specific plant carbohydrates, including fructan metabolites, during the switch to necrotrophic growth and reproduction. This switch coincided with increased expression of jasmonic acid biosynthesis genes and large-scale activation of other plant defense responses. Fungal genes encoding putative secondary metabolite clusters and secreted effector proteins were identified with distinct infection phase-specific expression patterns, although functional analysis suggested that many have overlapping/redundant functions in virulence. The pathogenic lifestyle of Z. tritici on wheat revealed through this study, involving initial defense suppression by a slow-growing extracellular and nutritionally limited pathogen followed by defense (hyper) activation during reproduction, reveals a subtle modification of the conceptual definition of hemibiotrophic plant infection.

The Wheat Mitogen-Activated Protein Kinases TaMPK3 and TaMPK6 Are Differentially Regulated at Multiple Levels during Compatible Disease Interactions with Mycosphaerella graminicola

Many race- or isolate-specific disease resistance responses of plants toward pathogens (incompatible interactions) invoke hypersensitive response (HR)-like programmed cell death (PCD) and the coordinated activation of mitogen-activated protein kinases homologous with Arabidopsis (Arabidopsis thaliana) AtMPK6 and AtMPK3 (or tobacco [Nicotiana tabacum] SIPK and WIPK), respectively. Resistance of wheat (Triticum aestivum) leaves to the necrotrophic fungal pathogen Mycosphaerella graminicola can also operate at an isolate/cultivar-specific level. We confirm here that resistance is achieved without any sign of HR-like PCD during the incompatible interaction. Instead, PCD is strictly associated with the compatible interaction and is triggered during disease symptom expression. A strong transcriptional activation of TaMPK3, the wheat homolog of Arabidopsis AtMPK3, was observed immediately preceding PCD and symptom development in the compatible interaction. Generation and use of TaMPK3- and TaMPK6-specific antibodies on western blots and in coupled immunoprecipitation-protein kinase assays demonstrated that the TaMPK3 protein also accumulated, and was subsequently posttranslationally activated, during the compatible interaction in parallel to PCD. In contrast, no increase in expression, protein levels, or posttranslational activation of TaMPK6 was observed at any stage of either compatible or incompatible interactions. However, the protein levels of TaMPK6 became markedly reduced during the compatible interaction coincident with the onset of TaMPK3 protein accumulation. These data highlight the emerging similarity between the signaling pathways triggered in a host plant during successful infection by a necrotrophic fungal pathogen and the resistance responses normally effective against biotrophs.

Molecular characterization and functional analysis of MgNLP, the sole NPP1 domain-containing protein, from the fungal wheat leaf pathogen Mycosphaerella graminicola.

Analysis of the fully sequenced genome of the wheat leaf-specific fungal pathogen Mycosphaerella graminicola identified only a single gene encoding a member of the necrosis- and ethylene-inducing peptide 1 (Nep1)-like protein family (NLP). NLP proteins have frequently been shown to trigger cell death and the activation of defense signaling reactions in dicotyledonous plants. However, complete loss-of-function reverse genetics analyses for their importance in the virulence of eukaryotic plant pathogens are generally lacking. Real-time quantitative polymerase chain reaction on MgNLP demonstrated the gene to be specifically expressed in planta. Peak expression was observed during the immediate presymptomatic phase of colonization of a susceptible host genotype. This was followed by a dramatic decrease during disease lesion formation which, in this system, exhibits characteristics of host programmed cell death (PCD). No comparable peak in transcript levels was seen during an incompatible interaction with a host genotype exhibiting gene-for-gene-based disease resistance. Heterologously expressed MgNLP protein induced necrotic cell death and the activation of defense-related genes when infiltrated into Arabidopsis leaves but not in leaves of a susceptible wheat genotype. MgNLP infiltration also failed to stimulate wheat mitogen-activated protein kinase activities. Finally, targeted deletion of M. graminicola MgNLP caused no detectable reduction in plant pathogenicity or virulence, suggesting that this protein is not a major virulence determinant during fungal infection of its host plant. To our knowledge, this represents the first complete loss-of-function analysis of NLP in a eukaryotic plant pathogen and we discuss our findings in the context of possible functions for NLP in pathogens which only infect monocotyledonous plants.

Mycosphaerella graminicola LysM Effector-Mediated Stealth Pathogenesis Subverts Recognition Through Both CERK1 and CEBiP Homologues in Wheat

Fungal cell-wall chitin is a well-recognized pathogen-associated molecular pattern. Recognition of chitin in plants by pattern recognition receptors activates pathogen-triggered immunity (PTI). In Arabidopsis, this process is mediated by a plasma membrane receptor kinase, CERK1, whereas in rice, a receptor-like protein, CEBiP, in addition to CERK1 is required. Secreted chitin-binding lysin motif (LysM) containing fungal effector proteins, such as Ecp6 from the biotrophic fungus Cladosporium fulvum, have been reported to interfere with PTI. Here, we identified wheat homologues of CERK1 and CEBiP and investigated their role in the interaction with the nonbiotrophic pathogen of wheat Mycosphaerella graminicola (synonym Zymoseptoria tritici). We show that silencing of either CERK1 or CEBiP in wheat, using Barley stripe mosaic virus-mediated virus-induced gene silencing, is sufficient in allowing leaf colonization by the normally nonpathogenic M. graminicola Mg3LysM (homologue of Ecp6) deletion mutant, while the Mg1LysM deletion mutant was fully pathogenic toward both silenced and wild-type wheat leaves. These data indicate that Mg3LysM is important for fungal evasion of PTI in wheat leaf tissue and that both CERK1 and CEBiP are required for activation of chitin-induced defenses, a feature conserved between rice and wheat, and perhaps, also in other cereal species.

Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen

In higher plants, sexual reproduction involves interactions between pollen and pistil. A key mechanism to prevent inbreeding is self-incompatibility through rejection of incompatible (‘self’) pollen. In Papaver rhoeas, S proteins encoded by the stigma interact with incompatible pollen, triggering a Ca2+-dependent signalling network resulting in pollen tube inhibition and programmed cell death. The cytosolic phosphoprotein p26.1, which has been identified in incompatible pollen, shows rapid, self-incompatibility-induced Ca2+-dependent hyperphosphorylation in vivo. Here we show that p26.1 comprises two proteins, Pr-p26.1a and Pr-p26.1b, which are soluble inorganic pyrophosphatases (sPPases). These proteins have classic Mg2+-dependent sPPase activity, which is inhibited by Ca2+, and unexpectedly can be phosphorylated in vitro. We show that phosphorylation inhibits sPPase activity, establishing a previously unknown mechanism for regulating eukaryotic sPPases. Reduced sPPase activity is predicted to result in the inhibition of many biosynthetic pathways, suggesting that there may be additional mechanisms of self-incompatibility-mediated pollen tube inhibition. We provide evidence that sPPases are required for growth and that self-incompatibility results in an increase in inorganic pyrophosphate, implying a functional role for Pr-p26.1.