Stomatal penetration: the cornerstone of plant resistance to the fungal pathogen Zymoseptoria tritici.

BACKGROUND: Septoria tritici blotch (STB), caused by the foliar fungus Zymoseptoria tritici, is one of the most damaging disease of wheat in Europe. Genetic resistance against this fungus relies on different types of resistance from non-host resistance (NHR) and host species specific resistance (HSSR) to host resistance mediated by quantitative trait loci (QTLs) or major resistance genes (Stb). Characterizing the diversity of theses resistances is of great importance for breeding wheat cultivars with efficient and durable resistance. While the functional mechanisms underlying these resistance types are not well understood, increasing piece of evidence suggest that fungus stomatal penetration and early establishment in the apoplast are both crucial for the outcome of some interactions between Z. tritici and plants. To validate and extend these previous observations, we conducted quantitative comparative phenotypical and cytological analyses of the infection process corresponding to 22 different interactions between plant species and Z. tritici isolates. These interactions included four major bread wheat Stb genes, four bread wheat accessions with contrasting quantitative resistance, two species resistant to Z. tritici isolates from bread wheat (HSSR) and four plant species resistant to all Z. tritici isolates (NHR). RESULTS: Infiltration of Z. tritici spores into plant leaves allowed the partial bypass of all bread wheat resistances and durum wheat resistance, but not resistances from other plants species. Quantitative comparative cytological analysis showed that in the non-grass plant Nicotiana benthamiana, Z. tritici was stopped before stomatal penetration. By contrast, in all resistant grass plants, Z. tritici was stopped, at least partly, during stomatal penetration. The intensity of this early plant control process varied depending on resistance types, quantitative resistances being the least effective. These analyses also demonstrated that Stb-mediated resistances, HSSR and NHR, but not quantitative resistances, relied on the strong growth inhibition of the few Z. tritici penetrating hyphae at their entry point in the sub-stomatal cavity. CONCLUSIONS: In addition to furnishing a robust quantitative cytological assessment system, our study uncovered three stopping patterns of Z. tritici by plant resistances. Stomatal resistance was found important for most resistances to Z. tritici, independently of its type (Stb, HSSR, NHR). These results provided a basis for the functional analysis of wheat resistance to Z. tritici and its improvement.

Sas3-mediated histone acetylation regulates effector gene activation in a fungal plant pathogen.

IMPORTANCE: Pathogen infections require the production of effectors that enable host colonization. Effectors have diverse functions and are only expressed at certain stages of the infection cycle. Thus, effector genes are tightly regulated by several mechanisms, including chromatin remodeling. Here, we investigate the role of histone acetylation in effector gene activation in the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that lysine acetyltransferases (KATs) are essential for the spatiotemporal regulation of effector genes. We show that the KAT Sas3 is involved in leaf symptom development and pycnidia formation. Importantly, our results indicate that Sas3 controls histone acetylation of effector loci and is a regulator of effector gene activation during stomatal penetration. Overall, our work demonstrates the key role of histone acetylation in regulating gene expression associated with plant infection.

Chitosan induces differential transcript usage of chitosanase 3 encoding gene (csn3) in the biocontrol fungus Pochonia chlamydosporia 123.

BACKGROUND: Pochonia chlamydosporia is an endophytic fungus used for nematode biocontrol that employs its cellular and molecular machinery to degrade the nematode egg-shell. Chitosanases, among other enzymes, are involved in this process. In this study, we improve the genome sequence assembly of P. chlamydosporia 123, by utilizing long Pacific Biosciences (PacBio) sequence reads. Combining this improved genome assembly with previous RNA-seq data revealed alternative isoforms of a chitosanase in the presence of chitosan. This study could open new insights into understanding fungal resistance to chitosan and root-knot nematode (RKN) egg infection processes. RESULTS: The P. chlamydosporia 123 genome sequence assembly has been updated using long-read PacBio sequencing and now includes 12,810 predicted protein-coding genes. Compared with the previous assembly based on short reads, there are 701 newly annotated genes, and 69 previous genes are now split. Eight of the new genes were differentially expressed in fungus interactions with Meloidogyne javanica eggs or chitosan. A survey of the RNA-seq data revealed alternative splicing in the csn3 gene that encodes a chitosanase, with four putative splicing variants: csn3_v1, csn3_v2, csn3_v3 and csn3_v4. When P. chlamydosporia is treated with 0.1 mg·mL- 1 chitosan for 4 days, csn3 is expressed 10-fold compared with untreated controls. Furthermore, the relative abundances of each of the four transcripts are different in chitosan treatment compared with controls. In controls, the abundances of each transcript are nil, 32, 55, and 12% for isoforms csn3_v1, csn3_v2, csn3_v3 and csn3_v4 respectively. Conversely, in chitosan-treated P. chlamydosporia, the abundances are respectively 80, 15%, 2-3%, 2-3%. Since isoform csn3_v1 is expressed with chitosan only, the putatively encoded enzyme is probably induced and likely important for chitosan degradation. CONCLUSIONS: Alternative splicing events have been discovered and described in the chitosanase 3 encoding gene from P. chlamydosporia 123. Gene csn3 takes part in RKN parasitism process and chitosan enhances its expression. The isoform csn3_v1 would be related to the degradation of this polymer in bulk form, while other isoforms may be related to the degradation of chitosan in the nematode egg-shell.

Chitosan and nematophagous fungi for sustainable management of nematode pests.

Plants are exposed to large number of threats caused by herbivores and pathogens which cause important losses on crops. Plant pathogens such as nematodes can cause severe damage and losses in food security crops worldwide. Chemical pesticides were extendedly used for nematode management. However, due to their adverse effects on human health and the environment, they are now facing strong limitations by regulatory organisations such as EFSA (European Food Safety Authority). Therefore, there is an urgent need for alternative and efficient control measures, such as biological control agents or bio-based plant protection compounds. In this scenario, chitosan, a non-toxic polymer obtained from seafood waste mainly, is becoming increasingly important. Chitosan is the N-deacetylated form of chitin. Chitosan is effective in the control of plant pests and diseases. It also induces plants defence mechanisms. Chitosan is also compatible with some biocontrol microorganisms mainly entomopathogenic and nematophagous fungi. Some of them are antagonists of nematode pests of plants and animals. The nematophagous biocontrol fungus Pochonia chlamydosporia has been widely studied for sustainable management of nematodes affecting economically important crops and for its capability to grow with chitosan as only nutrient source. This fungus infects nematode eggs using hyphal tips and appressoria. Pochonia chlamydosporia also colonizes plant roots endophytically, stimulating plant defences by induction of salicylic and jasmonic acid biosynthesis and favours plant growth and development. Therefore, the combined use of chitosan and nematophagous fungi could be a novel strategy for the biological control of nematodes and other root pathogens of food security crops.

Putative LysM Effectors Contribute to Fungal Lifestyle.

Fungal LysM effector proteins can dampen plant host-defence responses, protecting hyphae from plant chitinases, but little is known on these effectors from nonpathogenic fungal endophytes. We found four putative LysM effectors in the genome of the endophytic nematophagous fungus Pochonia chlamydosporia (Pc123). All four genes encoding putative LysM effectors are expressed constitutively by the fungus. Additionally, the gene encoding Lys1-the smallest one-is the most expressed in banana roots colonised by the fungus. Pc123 Lys1, 2 and 4 display high homology with those of other strains of the fungus and phylogenetically close entomopathogenic fungi. However, Pc123 Lys3 displays low homology with other fungi, but some similarities are found in saprophytes. This suggests evolutionary divergence in Pc123 LysM effectors. Additionally, molecular docking shows that the NAcGl binding sites of Pc123 Lys 2, 3 and 4 are adjacent to an alpha helix. Putative LysM effectors from fungal endophytes, such as Pc123, differ from those of plant pathogenic fungi. LysM motifs from endophytic fungi show clear conservation of cysteines in Positions 13, 51 and 63, unlike those of plant pathogens. LysM effectors could therefore be associated with the lifestyle of a fungus and give us a clue of how organisms could behave in different environments.

Chitosan modulates Pochonia chlamydosporia gene expression during nematode egg parasitism.

Climate change makes plant-parasitic nematodes (PPN) an increasing threat to commercial crops. PPN can be managed sustainably by the biocontrol fungus Pochonia chlamydosporia (Pc). Chitosan generated from chitin deacetylation enhances PPN parasitism by Pc. In this work, we investigate the molecular mechanisms of Pc for chitosan resistance and root-knot nematode (RKN) parasitism, using transcriptomics. Chitosan and RKN modify the expression of Pc genes, mainly those involved in oxidation-reduction processes. Both agents significantly modify the expression of genes associated to 113 GO terms and 180 Pc genes. Genes encoding putative glycoproteins (Pc adhesives) to nematode eggshell, as well as genes involved in redox, carbohydrate and lipid metabolism trigger the response to chitosan. We identify genes expressed in both the parasitic and endophytic phases of the Pc lifecycle; these include proteases, chitosanases and transcription factors. Using the Pathogen-Host Interaction database (PHI-base), our previous RNA-seq data and RT-PCR of Pc colonizing banana we have investigated genes expressed both in the parasitic and endophytic phases of Pc lifecycle.

Chitosan Induces Plant Hormones and Defenses in Tomato Root Exudates.

In this work, we use electrophysiological and metabolomic tools to determine the role of chitosan as plant defense elicitor in soil for preventing or manage root pests and diseases sustainably. Root exudates include a wide variety of molecules that plants and root microbiota use to communicate in the rhizosphere. Tomato plants were treated with chitosan. Root exudates from tomato plants were analyzed at 3, 10, 20, and 30 days after planting (dap). We found, using high performance liquid chromatography (HPLC) and excitation emission matrix (EEM) fluorescence, that chitosan induces plant hormones, lipid signaling and defense compounds in tomato root exudates, including phenolics. High doses of chitosan induce membrane depolarization and affect membrane integrity. 1H-NMR showed the dynamic of exudation, detecting the largest number of signals in 20 dap root exudates. Root exudates from plants irrigated with chitosan inhibit ca. twofold growth kinetics of the tomato root parasitic fungus Fusarium oxysporum f. sp. radicis-lycopersici. and reduced ca. 1.5-fold egg hatching of the root-knot nematode Meloidogyne javanica.

Molecular Mechanisms of Chitosan Interactions with Fungi and Plants.

Chitosan is a versatile compound with multiple biotechnological applications. This polymer inhibits clinically important human fungal pathogens under the same carbon and nitrogen status as in blood. Chitosan permeabilises their high-fluidity plasma membrane and increases production of intracellular oxygen species (ROS). Conversely, chitosan is compatible with mammalian cell lines as well as with biocontrol fungi (BCF). BCF resistant to chitosan have low-fluidity membranes and high glucan/chitin ratios in their cell walls. Recent studies illustrate molecular and physiological basis of chitosan-root interactions. Chitosan induces auxin accumulation in Arabidopsis roots. This polymer causes overexpression of tryptophan-dependent auxin biosynthesis pathway. It also blocks auxin translocation in roots. Chitosan is a plant defense modulator. Endophytes and fungal pathogens evade plant immunity converting chitin into chitosan. LysM effectors shield chitin and protect fungal cell walls from plant chitinases. These enzymes together with fungal chitin deacetylases, chitosanases and effectors play determinant roles during fungal colonization of plants. This review describes chitosan mode of action (cell and gene targets) in fungi and plants. This knowledge will help to develop chitosan for agrobiotechnological and medical applications.

Genome and secretome analysis of Pochonia chlamydosporia provide new insight into egg-parasitic mechanisms.

Pochonia chlamydosporia infects eggs and females of economically important plant-parasitic nematodes. The fungal isolates parasitizing different nematodes are genetically distinct. To understand their intraspecific genetic differentiation, parasitic mechanisms, and adaptive evolution, we assembled seven putative chromosomes of P. chlamydosporia strain 170 isolated from root-knot nematode eggs (~44 Mb, including 7.19% of transposable elements) and compared them with the genome of the strain 123 (~41 Mb) isolated from cereal cyst nematode. We focus on secretomes of the fungus, which play important roles in pathogenicity and fungus-host/environment interactions, and identified 1,750 secreted proteins, with a high proportion of carboxypeptidases, subtilisins, and chitinases. We analyzed the phylogenies of these genes and predicted new pathogenic molecules. By comparative transcriptome analysis, we found that secreted proteins involved in responses to nutrient stress are mainly comprised of proteases and glycoside hydrolases. Moreover, 32 secreted proteins undergoing positive selection and 71 duplicated gene pairs encoding secreted proteins are identified. Two duplicated pairs encoding secreted glycosyl hydrolases (GH30), which may be related to fungal endophytic process and lost in many insect-pathogenic fungi but exist in nematophagous fungi, are putatively acquired from bacteria by horizontal gene transfer. The results help understanding genetic origins and evolution of parasitism-related genes.