Effect of Pochonia chlamydosporia Endophytism and Meloidogyne incognita Parasitism on WRKYs and Defense Gene Expression in Tomato Roots.

The expression of WRKY transcription factors and plant defense-related genes was studied in the roots of Apulian tomato (Solanum lycopersicum) cv Regina di Fasano (accessions MRT and PLZ) endophytically colonized by Pochonia chlamydosporia and parasitized or not by the root-knot nematode (RKN) Meloidogyne incognita. The effect on plant growth, nematode parasitism and histological aspect of the interaction were considered. The association of P. chlamydosporia with RKN-parasitized MRT plants increased the total biomass and shoot fresh weight in comparison with healthy plants and with those only parasitized by RKN, without the endophyte. However, the PLZ accession showed no significant difference in the observed biometric parameters. The number of RKN-induced galls per plant was not affected by endophytism eight days after inoculation. No histological changes were observed in the nematode feeding sites in the presence of the fungus. Gene expression analysis showed an accession-specific response to P. chlamydosporia with differential activation of WRKY-related genes. No significant change was found for WRKY76 expression in nematode-parasitized plants compared with control roots, confirming cultivar susceptibility. Data indicate genotype-specific responses of the WRKY genes to parasitism examined in roots with nematodes and/or endophytic P. chlamydosporia. At 25 days post-inoculation with P. chlamydosporia, no significant difference was observed in the expression of defense-related genes in both accessions, suggesting that salicylic acid (SA) (PAL and PR1) and jasmonate (JA) related genes (Pin II) are not active during endophytism.

Water Stress Differentially Modulates the Expression of Tomato Cell Wall Metabolism-Related Genes in Meloidogyne incognita Feeding Sites.

Microscopic observations and transcriptomic RNA-Seq analyses were applied to investigate the effect of water stress during the formation of tomato galls formation 1 and 2 weeks after inoculation with the root-knot nematode Meloidogyne incognita. Water stress affected root growth and the nematode ability to mount an efficient parasitism. The effects of water stress on the feeding site development were already observed at 1 week after nematode inoculation, with smaller giant cells, delayed development, and thinner cell walls. These features suggested changes in the expression levels of genes involved in the feeding site formation and maintenance. Gene Ontology (GO) enrichment and expression patterns were used to characterize differentially expressed genes. Water stress modified the expression profile of genes involved in the synthesis, degradation, and remodeling of the cell wall during the development of nematode feeding site. A comparison of gene expression with unstressed galls revealed that water stress intensified the up or downregulation of most genes. However, it particularly influenced the expression pattern of expansin A11 (Solyc04g081870.4.1), expansin-like B1(Solyc08g077910.3.1), a pectin acetylesterase (Solyc08g005800.4.1), and the pectin methylesterase pmeu1 (Solyc03g123630.4.1) which were upregulated in unstressed galls and repressed by water stress, at both sampling times. The expression of most members of the genes involved in cell wall metabolism, i.e., those coding for Csl, fasciclin, and COBRA proteins, were negatively influenced. Interestingly, alteration in the expression profiles of most dirigent protein genes (DIRs) and upregulation of five gene coding for Casparian strip domain protein (CASP)-like proteins were found. Gene expression analysis of galls from water stressed plants allowed us to better understand the molecular basis of M. incognita parasitism in tomato. Specific genes, including those involved in regulation of cellulose synthesis and lignification process, require further study to develop defense strategies against root-knot nematodes.

Root Endophytism by Pochonia chlamydosporia Affects Defense-Gene Expression in Leaves of Monocot and Dicot Hosts under Multiple Biotic Interactions.

A study was carried out on the effect of the root endophytic fungus Pochonia chlamydosporia on plant systemic signal of defense related genes during fungal or nematode parasitism. Different biotic stress factors were examined, inoculating roots of dicot and monocot hosts with the endophyte, and measuring the expression of defense genes in leaves. A first greenhouse assay was carried out on expression of PAL, PIN II, PR1 and LOX D in leaves of tomato cv Tondino inoculated with Phytophthora infestans (CBS 120920), inoculating or not the roots of infected plants with P. chlamydosporia DSM 26985. In a second assay, plants of banana (Musa acuminata cv Grand Naine) were artificially infected with Fusarium oxysporum f. sp. cubense Tropical race 4 (TR4) and inoculated or not with DSM 26985. In a further experiment, banana plants were inoculated or not with P. chlamydosporia plus juveniles of the root knot nematode (RKN) Meloidogyne incognita. A similar assay was also carried out in vitro with adults and juveniles of the lesion nematode Pratylenchus goodeyi. Differential expression of the defense genes examined was observed for all plant-stress associations, indicative of early, upward systemic signals induced by the endophyte. Changes in expression profiles included a 5-fold down-regulation of PIN II at 2 dai in leaves of tomato plants treated with P. infestans and/or P. chlamydosporia, and the up-regulation of PAL by the endophyte alone, at 2 and 7 dai. In the TR4 assay, PR1 was significantly up-regulated at 7 dai in banana leaves, but only in the P. chlamydosporia treated plants. At 10 dai, PIN II expression was significantly higher in leaves of plants inoculated only with TR4. The banana-RKN assay showed a PR1 expression significantly higher than controls at 4 and 7 dai in plants inoculated with P. chlamydosporia alone, and a down-regulation at 4 dai in leaves of plants also inoculated with RKN, with a PR1 differential up-regulation at 10 dai. Pratylenchus goodeyi down-regulated PIN at 21 dai, with or without the endophyte, as well as PAL but only in presence of P. chlamydosporia. When inoculated alone, the endophyte up-regulated PR1 and LOX. The gene expression patterns observed in leaves suggest specific and time-dependent relationships linking host plants and P. chlamydosporia in presence of biotic stress factors, functional to a systemic, although complex, activation of defense genes.

Omics applications: towards a sustainable protection of tomato.

Transcriptome data and gene expression analysis have a huge potential in the study of multiple relationships involving plants, pathogens, and pests, including the interactions with beneficial microorganisms such as endophytes or other functional groups. Next-generation sequencing (NGS) and other recent long-read-based sequencing approaches (i.e., nanopore and others) provide unprecedented tools allowing the fast identification of plant information processing systems, in situ and in real time, fundamental for crop management and pest regulation. Other -omics approaches such as metagenomics and metatranscriptomics allow high-resolution insights on the rhizosphere ecology. They may highlight key factors affecting belowground biodiversity or processes, modulating the expression of stress-responsive pathways. The application of miRNAs and other small RNAs is a relatively new field of application, with enormous potential for the selective activation of defense pathways. However, limitations concerning the stability of the RNA molecules and their effective delivery must be overcome.

Time-dependent effects of Pochonia chlamydosporia endophytism on gene expression profiles of colonized tomato roots.

A transcriptome analysis was produced from tomato roots inoculated with the hyphomycete Pochonia chlamydosporia at three different times. Gene expression data were also yielded from fungus grown in vitro or endophytic. A next-generation sequencing (NGS) and network analysis approach were applied. We identified 3.676 differentially expressed tomato genes (DEG), highlighting a core of 93 transcripts commonly down- or upregulated at every time point, shedding light on endophytism process. Functional categories related to plant information-processing system, which recognizes, percepts, and transmits signals, were associated with gene upregulated early in time, with higher representations in processes such as plant defense regulation later in time. Network analysis of a DEG subset showed dominance of MAP kinase hubs in the uninoculated control samples, replaced by an increased centrality of WRKY transcription factor and ETR-ethylene response factor genes in the colonized roots. Fungus genes expressed during progression of plant colonization, therefore related to the host colonization process or endophytism persistence, were also identified. Data provided a high-resolution insight on tomato transcriptome changes as induced by endophytism, highlighting a specific modulation of stress-responsive transcripts, related to a selective activation of defense pathways, likely required by the fungus to establish a persistent endophytic lifestyle.

Transcriptomic Responses to Water Deficit and Nematode Infection in Mycorrhizal Tomato Roots.

Climate changes include the intensification of drought in many parts of the world, increasing its frequency, severity and duration. However, under natural conditions, environmental stresses do not occur alone, and, in addition, more stressed plants may become more susceptible to attacks by pests and pathogens. Studies on the impact of the arbuscular mycorrhizal (AM) symbiosis on tomato response to water deficit showed that several drought-responsive genes are differentially regulated in AM-colonized tomato plants (roots and leaves) during water deficit. To date, global changes in mycorrhizal tomato root transcripts under water stress conditions have not been yet investigated. Here, changes in root transcriptome in the presence of an AM fungus, with or without water stress (WS) application, have been evaluated in a commercial tomato cultivar already investigated for the water stress response during AM symbiosis. Since root-knot nematodes (RKNs, Meloidogyne incognita) are obligate endoparasites and cause severe yield losses in tomato, the impact of the AM fungal colonization on RKN infection at 7 days post-inoculation was also evaluated. Results offer new information about the response to AM symbiosis, highlighting a functional redundancy for several tomato gene families, as well as on the tomato and fungal genes involved in WS response during symbiosis, underlying the role of the AM fungus. Changes in the expression of tomato genes related to nematode infection during AM symbiosis highlight a role of AM colonization in triggering defense responses against RKN in tomato. Overall, new datasets on the tomato response to an abiotic and biotic stress during AM symbiosis have been obtained, providing useful data for further researches.

Identification of tomato miRNAs responsive to root colonization by endophytic Pochonia chlamydosporia.

The molecular mechanisms active during the endophytic phase of the fungus Pochonia chlamydosporia are still poorly understood. In particular, few data are available on the links between the endophyte and the root response, as modulated by noncoding small RNAs. In this study, we describe the microRNAs (miRNAs) that are differentially expressed (DE) in the roots of tomato, colonized by P. chlamydosporia. A genome-wide NGS expression profiling of small RNAs in roots, either colonized or not by the fungus, showed 26 miRNAs upregulated in inoculated roots. Their predicted target genes are involved in the plant information processing system, which recognizes, percepts, and transmits signals, with higher representations in processes such as apoptosis and plant defense regulation. RNAseq data showed that predicted miRNA target genes were downregulated in tomato roots after 4, 7, 10, and 21 days post P. chlamydosporia inoculation. The differential expression of four miRNAs was further validated using qPCR analysis. The P. chlamydosporia endophytic lifestyle in tomato roots included an intricate network of miRNAs and targets. Data provide a first platform of DE tomato miRNAs after P. chlamydosporia colonization. They indicated that several miRNAs are involved in the host response to the fungus, playing important roles for its recognition as a symbiotic microorganism, allowing endophytism by modulating the host defense reaction. Data also indicated that endophytism affects tRNA fragmentation. This is the first study on miRNAs induced by P. chlamydosporia endophytism and related development regulation effects in Solanum lycopersicum.