Specific tissue proteins 1 and 6 are involved in root biology during normal development and under symbiotic and pathogenic interactions in Medicago truncatula.

MAIN CONCLUSION: ST1 and ST6 are possibly involved in primary and lateral root and symbiotic nodule development, but only ST6 participates in the interaction with hemibiotrophic fungi. Specific tissue (ST) proteins have been shown to be involved in several processes related to plant nutritional status, development, and responses to biotic agents. In particular, ST1 and ST6 are mainly expressed in roots throughout plant development. Here, we analyze where and how the expression of the genes encoding both proteins are modulated in the legume model plant Medicago truncatula in response to the plant developmental program, nodulation induced by a beneficial nitrogen-fixing bacterium (Sinorhizobium meliloti) and the defense response triggered by a pathogenic hemibiotrophic fungus (Fusarium oxysporum). Gene expression results show that ST1 and ST6 participate in the vasculature development of both primary and lateral roots, although only ST6 is related to meristem activity. ST1 and ST6 clearly display different roles in the biotic interactions analyzed, where ST1 is activated in response to a N2-fixing bacterium and ST6 is up-regulated after inoculation with F. oxysporum. The role of ST1 and ST6 in the nodulation process may be related to nodule organogenesis rather than to the establishment of the interaction itself, and an increase in ST6 correlates with the activation of the salicylic acid signaling pathway during the infection and colonization processes. These results further support the role of ST6 in response to hemibiotrophic fungi. This research contributes to the understanding of the complex network that controls root biology and strengthens the idea that ST proteins are involved in several processes such as primary and lateral root development, nodule organogenesis, and the plant-microbe interaction.

Bcmimp1, a Botrytis cinerea Gene Transiently Expressed in planta, Encodes a Mitochondrial Protein.

Botrytis cinerea is a widespread necrotrophic fungus which infects more than 200 plant species. In an attempt to characterize the physiological status of the fungus in planta and to identify genetic factors contributing to its ability to infect the host cells, a differential gene expression analysis during the interaction B. cinerea-tomato was carried out. Gene Bcmimp1 codes for a mRNA detected by differential display in the course of this analysis. During the interaction with the host, it shows a transient expression pattern with maximal expression levels during the colonization and maceration of the infected tissues. Bioinformatic analysis suggested that BCMIMP1 is an integral membrane protein located in the mitochondrial inner membrane. Co-localization experiments with a BCMIMP1-GFP fusion protein confirmed that the protein is targeted to the mitochondria. ΔBcmimp1 mutants do not show obvious phenotypic differences during saprophytic growth and their infection ability was unaltered as compared to the wild-type. Interestingly, the mutants produced increased levels of reactive oxygen species, likely as a consequence of disturbed mitochondrial function. Although Bcmimp1 expression is enhanced in planta it cannot be considered a pathogenicity factor.

A Fungal Effector With Host Nuclear Localization and DNA-Binding Properties Is Required for Maize Anthracnose Development.

Plant pathogens have the capacity to manipulate the host immune system through the secretion of effectors. We identified 27 putative effector proteins encoded in the genome of the maize anthracnose pathogen Colletotrichum graminicola that are likely to target the host's nucleus, as they simultaneously contain sequence signatures for secretion and nuclear localization. We functionally characterized one protein, identified as CgEP1. This protein is synthesized during the early stages of disease development and is necessary for anthracnose development in maize leaves, stems, and roots. Genetic, molecular, and biochemical studies confirmed that this effector targets the host's nucleus and defines a novel class of double-stranded DNA-binding protein. We show that CgEP1 arose from a gene duplication in an ancestor of a lineage of monocot-infecting Colletotrichum spp. and has undergone an intense evolution process, with evidence for episodes of positive selection. We detected CgEP1 homologs in several species of a grass-infecting lineage of Colletotrichum spp., suggesting that its function may be conserved across a large number of anthracnose pathogens. Our results demonstrate that effectors targeted to the host nucleus may be key elements for disease development and aid in the understanding of the genetic basis of anthracnose development in maize plants.

A highly conserved metalloprotease effector enhances virulence in the maize anthracnose fungus Colletotrichum graminicola.

Colletotrichum graminicola causes maize anthracnose, an agronomically important disease with a worldwide distribution. We have identified a fungalysin metalloprotease (Cgfl) with a role in virulence. Transcriptional profiling experiments and live cell imaging show that Cgfl is specifically expressed during the biotrophic stage of infection. To determine whether Cgfl has a role in virulence, we obtained null mutants lacking Cgfl and performed pathogenicity and live microscopy assays. The appressorium morphology of the null mutants is normal, but they exhibit delayed development during the infection process on maize leaves and roots, showing that Cgfl has a role in virulence. In vitro chitinase activity assays of leaves infected with wild-type and null mutant strains show that, in the absence of Cgfl, maize leaves exhibit increased chitinase activity. Phylogenetic analyses show that Cgfl is highly conserved in fungi. Similarity searches, phylogenetic analysis and transcriptional profiling show that C. graminicola encodes two LysM domain-containing homologues of Ecp6, suggesting that this fungus employs both Cgfl-mediated and LysM protein-mediated strategies to control chitin signalling.

Plant defense mechanisms are activated during biotrophic and necrotrophic development of Colletotricum graminicola in maize.

Hemibiotrophic plant pathogens first establish a biotrophic interaction with the host plant and later switch to a destructive necrotrophic lifestyle. Studies of biotrophic pathogens have shown that they actively suppress plant defenses after an initial microbe-associated molecular pattern-triggered activation. In contrast, studies of the hemibiotrophs suggest that they do not suppress plant defenses during the biotrophic phase, indicating that while there are similarities between the biotrophic phase of hemibiotrophs and biotrophic pathogens, the two lifestyles are not analogous. We performed transcriptomic, histological, and biochemical studies of the early events during the infection of maize (Zea mays) with Colletotrichum graminicola, a model pathosystem for the study of hemibiotrophy. Time-course experiments revealed that mRNAs of several defense-related genes, reactive oxygen species, and antimicrobial compounds all begin to accumulate early in the infection process and continue to accumulate during the biotrophic stage. We also discovered the production of maize-derived vesicular bodies containing hydrogen peroxide targeting the fungal hyphae. We describe the fungal respiratory burst during host infection, paralleled by superoxide ion production in specific fungal cells during the transition from biotrophy to a necrotrophic lifestyle. We also identified several novel putative fungal effectors and studied their expression during anthracnose development in maize. Our results demonstrate a strong induction of defense mechanisms occurring in maize cells during C. graminicola infection, even during the biotrophic development of the pathogen. We hypothesize that the switch to necrotrophic growth enables the fungus to evade the effects of the plant immune system and allows for full fungal pathogenicity.