In-vitro and in-planta Botrytis cinerea Inoculation Assays for Tomato

Botrytis cinerea (B. cinerea) attacks many crops of economic importance, represents one of the most extensively studied necrotrophic pathogens. Inoculation of B. cinerea and phenotypic analysis of plant resistance are key procedures to investigate the mechanism of plant immunity. Here we describe a protocol for B. cinerea inoculation on medium and planta based on our study using the tomato-B. cinerea system

Stemphylium lycopersici Nep1-like Protein (NLP) Is a Key Virulence Factor in Tomato Gray Leaf Spot Disease

The fungus Stemphylium lycopersici (S. lycopersici) is an economically important plant pathogen that causes grey leaf spot disease in tomato. However, functional genomic studies in S. lycopersici are lacking, and the factors influencing its pathogenicity remain largely unknown. Here, we present the first example of genetic transformation and targeted gene replacement in S. lycopersici. We functionally analyzed the NLP gene, which encodes a necrosis- and ethylene-inducing peptide 1 (Nep1)-like protein (NLP). We found that targeted disruption of the NLP gene in S. lycopersici significantly compromised its virulence on tomato. Moreover, our data suggest that NLP affects S. lycopersici conidiospore production and weakly affects its adaptation to osmotic and oxidative stress. Interestingly, we found that NLP suppressed the production of reactive oxygen species (ROS) in tomato leaves during S. lycopersici infection. Further, expressing the fungal NLP in tomato resulted in constitutive transcription of immune-responsive genes and inhibited plant growth. Through gene manipulation, we demonstrated the function of NLP in S. lycopersici virulence and development. Our work provides a paradigm for functional genomics studies in a non-model fungal pathogen system

Characterization of Greenbeard Genes Involved in Long-Distance Kind Discrimination in a Microbial Eukaryote.

Microorganisms are capable of communication and cooperation to perform social activities. Cooperation can be enforced using kind discrimination mechanisms in which individuals preferentially help or punish others, depending on genetic relatedness only at certain loci. In the filamentous fungus Neurospora crassa, genetically identical asexual spores (germlings) communicate and fuse in a highly regulated process, which is associated with fitness benefits during colony establishment. Recognition and chemotropic interactions between isogenic germlings requires oscillation of the mitogen-activated protein kinase (MAPK) signal transduction protein complex (NRC-1, MEK-2, MAK-2, and the scaffold protein HAM-5) to specialized cell fusion structures termed conidial anastomosis tubes. Using a population of 110 wild N. crassa isolates, we investigated germling fusion between genetically unrelated individuals and discovered that chemotropic interactions are regulated by kind discrimination. Distinct communication groups were identified, in which germlings within one communication group interacted at high frequency, while germlings from different communication groups avoided each other. Bulk segregant analysis followed by whole genome resequencing identified three linked genes (doc-1, doc-2, and doc-3), which were associated with communication group phenotype. Alleles at doc-1, doc-2, and doc-3 fell into five haplotypes that showed transspecies polymorphism. Swapping doc-1 and doc-2 alleles from different communication group strains was necessary and sufficient to confer communication group affiliation. During chemotropic interactions, DOC-1 oscillated with MAK-2 to the tips of conidial anastomosis tubes, while DOC-2 was statically localized to the plasma membrane. Our data indicate that doc-1, doc-2, and doc-3 function as "greenbeard" genes, involved in mediating long-distance kind recognition that involves actively searching for one's own type, resulting in cooperation between non-genealogical relatives. Our findings serve as a basis for investigations into the mechanisms associated with attraction, fusion, and kind recognition in other eukaryotic species

Identification and characterization of LFD-2, a predicted fringe protein required for membrane integrity during cell fusion in neurospora crassa.

The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1 or Δlfd-1 germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, for late fusion defect-2) that showed a calcium-dependent cell lysis phenotype. lfd-2 encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter, fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion

Characterization of Greenbeard Genes Involved in Long-Distance Kind Discrimination in a Microbial Eukaryote.

Microorganisms are capable of communication and cooperation to perform social activities. Cooperation can be enforced using kind discrimination mechanisms in which individuals preferentially help or punish others, depending on genetic relatedness only at certain loci. In the filamentous fungus Neurospora crassa, genetically identical asexual spores (germlings) communicate and fuse in a highly regulated process, which is associated with fitness benefits during colony establishment. Recognition and chemotropic interactions between isogenic germlings requires oscillation of the mitogen-activated protein kinase (MAPK) signal transduction protein complex (NRC-1, MEK-2, MAK-2, and the scaffold protein HAM-5) to specialized cell fusion structures termed conidial anastomosis tubes. Using a population of 110 wild N. crassa isolates, we investigated germling fusion between genetically unrelated individuals and discovered that chemotropic interactions are regulated by kind discrimination. Distinct communication groups were identified, in which germlings within one communication group interacted at high frequency, while germlings from different communication groups avoided each other. Bulk segregant analysis followed by whole genome resequencing identified three linked genes (doc-1, doc-2, and doc-3), which were associated with communication group phenotype. Alleles at doc-1, doc-2, and doc-3 fell into five haplotypes that showed transspecies polymorphism. Swapping doc-1 and doc-2 alleles from different communication group strains was necessary and sufficient to confer communication group affiliation. During chemotropic interactions, DOC-1 oscillated with MAK-2 to the tips of conidial anastomosis tubes, while DOC-2 was statically localized to the plasma membrane. Our data indicate that doc-1, doc-2, and doc-3 function as "greenbeard" genes, involved in mediating long-distance kind recognition that involves actively searching for one's own type, resulting in cooperation between non-genealogical relatives. Our findings serve as a basis for investigations into the mechanisms associated with attraction, fusion, and kind recognition in other eukaryotic species

Identification of Allorecognition Loci in Neurospora crassa by Genomics and Evolutionary Approaches.

Understanding the genetic and molecular bases of the ability to distinguish self from nonself (allorecognition) and mechanisms underlying evolution of allorecognition systems is an important endeavor for understanding cases where it becomes dysfunctional, such as in autoimmune disorders. In filamentous fungi, allorecognition can result in vegetative or heterokaryon incompatibility, which is a type of programmed cell death that occurs following fusion of genetically different cells. Allorecognition is genetically controlled by het loci, with coexpression of any combination of incompatible alleles triggering vegetative incompatibility. Herein, we identified, characterized, and inferred the evolutionary history of candidate het loci in the filamentous fungus Neurospora crassa. As characterized het loci encode proteins carrying an HET domain, we annotated HET domain genes in 25 isolates from a natural population along with the N. crassa reference genome using resequencing data. Because allorecognition systems can be affected by frequency-dependent selection favoring rare alleles (i.e., balancing selection), we mined resequencing data for HET domain loci whose alleles displayed elevated levels of variability, excess of intermediate frequency alleles, and deep gene genealogies. From these analyses, 34 HET domain loci were identified as likely to be under balancing selection. Using transformation, incompatibility assays and genetic analyses, we determined that one of these candidates functioned as a het locus (het-e). The het-e locus has three divergent allelic groups that showed signatures of positive selection, intra- and intergroup recombination, and trans-species polymorphism. Our findings represent a compelling case of balancing selection functioning on multiple alleles across multiple loci potentially involved in allorecognition

Identification of allorecognition loci in neurospora crassa by genomics and evolutionary approaches

BGPI : équipe 5International audienceUnderstanding the genetic and molecular bases of the ability to distinguish self from nonself (allorecognition) and mechanisms underlying evolution of allorecognition systems is an important endeavor for understanding cases where it becomes dysfunctional, such as in autoimmune disorders. In filamentous fungi, allorecognition can result in vegetative or heterokaryon incompatibility, which is a type of programmed cell death that occurs following fusion of genetically different cells. Allorecognition is genetically controlled by het loci, with coexpression of any combination of incompatible alleles triggering vegetative incompatibility. Herein, we identified, characterized, and inferred the evolutionary history of candidate het loci in the filamentous fungus Neurospora crassa. As characterized het loci encode proteins carrying an HET domain, we annotated HET domain genes in 25 isolates from a natural population along with the N. crassa reference genome using resequencing data. Because allorecognition systems can be affected by frequency-dependent selection favoring rare alleles (i.e., balancing selection), we mined resequencing data for HET domain loci whose alleles displayed elevated levels of variability, excess of intermediate frequency alleles, and deep gene genealogies. From these analyses, 34 HET domain loci were identified as likely to be under balancing selection. Using transformation, incompatibility assays and genetic analyses, we determined that one of these candidates functioned as a het locus (het-e). The het-e locus has three divergent allelic groups that showed signatures of positive selection, intra-and intergroup recombination, and trans-species polymorphism. Our findings represent a compelling case of balancing selection functioning on multiple alleles across multiple loci potentially involved in allorecognition

Model for DOC-1 and DOC-2 function in long-distance kind discrimination.

<p>This model assumes that during conidial germination, <i>N</i>. <i>crassa</i> germlings release a ligand that acts as a signal for potential interaction partners. The ligand/receptor or other components of the recognition pathway might be modified and coded as “self” by DOC-1/DOC-2 of the sending cell. In the receiving cell, the ligand activates a receptor. The DOC-1/DOC-2 system of the receiving cell functions in quality control. If the signal passes quality control, oscillation of the assembled MAK-2 complex is enforced and the signal-receiving cell shows chemotropic interactions (top). If the signal does not pass quality control, the DOC-1/DOC-2 system of the receiving cell prevents enforcement of MAK-2 oscillation, and, therefore, the receiving cell does not respond to the presence of a potential fusion partner (bottom).</p