Valérie Pujade-Renaud

Expression of the HEV2.1 gene promoter in transgenic Hevea brasiliensis

Hevein is involved in the coagulation of rubber particles and is known to be highly expressed in latex cells from Hevea brasiliensis. As all HEVEIN genes are highly conserved in their transcribed region, a promoter functional analysis was carried out in transgenic rubber tree in order to evaluate the expression of HEV2.1, one member of the HEVEIN multigenic family. Three out of the 14 established independent transgenic lines carrying the HEV2.1::GUS construct were selected and set to regenerate plantlets. In situ hybridization of transgenic and wild-type plant tissues was respectively carried out with gusA and HEVEIN antisense mRNA probes. This showed co-localized-expression of the HEV2.1 gene with the overall HEVEIN gene expression. Laticifer-specific expression was observed in roots and stems, but not in leaves where HEVEIN genes were expressed in all cell types. Since HEV2.1::GUS callus lines responded significantly to light stimulation, up-regulation of the expression of HEV2.1 genes by light was suggested in leaves.

First characterization of endophytic Corynespora cassiicola isolates with variant cassiicolin genes recovered from rubber trees in Brazil

Corynespora cassiicola is the causal agent of Corynespora Leaf Fall (CLF) disease. CLF is one of the most important fungal diseases of rubber trees in Asia and Africa but disease outbreaks have not been reported in South America. Cassiicolin, a small cysteine-rich glycoprotein secreted by the pathogenic C. cassiicola isolate CCP, was previously identified as a potential disease effector in rubber tree. Recently, the cassiicolin-encoding gene (Cas1) was characterized and shown to be expressed in the early phase of infection. In this study, we investigated whether previously undetected strains of C. cassiicola are present in South American rubber plantations by examining the fungal endophyte population found in asymptomatic rubber tree leaves. Four isolates were identified as C. cassiicola. Genes encoding new forms of the cassiicolin precursor protein (Cas3 and Cas4) were identified from these isolates. Three of four isolates were able to induce symptoms on the cultivar they were isolated from in a detached leaf assay, with different kinetics and intensities. One isolate had the same pathogenicity profile as the pathogenic isolate CCP; the other two isolates developed symptoms late during the course of infection, suggesting saprotrophic capabilities. However, no Cas3 or Cas4 transcripts could be detected upon inoculation with the endophytic isolates, whereas the reference gene Cas1 was expressed upon inoculation with the CCP isolate. This work demonstrated that C. cassiicola is present in South America in an endophytic form and that it may evolve from an endophytic to a saprophytic or even potentially pathogenic life style.

Genetic Determinism of Sensitivity to Corynespora cassiicola Exudates in Rubber Tree (Hevea brasiliensis)

An indirect phenotyping method was developed in order to estimate the susceptibility of rubber tree clonal varieties to Corynespora Leaf Fall (CLF) disease caused by the ascomycete Corynespora cassiicola. This method consists in quantifying the impact of fungal exudates on detached leaves by measuring the induced electrolyte leakage (EL%). The tested exudates were either crude culture filtrates from diverse C. cassiicola isolates or the purified cassiicolin (Cas1), a small secreted effector protein produced by the aggressive isolate CCP. The test was found to be quantitative, with the EL% response proportional to toxin concentration. For eight clones tested with two aggressive isolates, the EL% response to the filtrates positively correlated to the response induced by conidial inoculation. The toxicity test applied to 18 clones using 13 toxinic treatments evidenced an important variability among clones and treatments, with a significant additional clone x treatment interaction effect. A genetic linkage map was built using 306 microsatellite markers, from the F1 population of the PB260 x RRIM600 family. Phenotyping of the population for sensitivity to the purified Cas1 effector and to culture filtrates from seven C. cassiicola isolates revealed a polygenic determinism, with six QTL detected on five chromosomes and percentages of explained phenotypic variance varying from 11 to 17%. Two common QTL were identified for the CCP filtrate and the purified cassiicolin, suggesting that Cas1 may be the main effector of CCP filtrate toxicity. The CCP filtrate clearly contrasted with all other filtrates. The toxicity test based on Electrolyte Leakage Measurement offers the opportunity to assess the sensitivity of rubber genotypes to C. cassiicola exudates or purified effectors for genetic investigations and early selection, without risk of spreading the fungus in plantations. However, the power of this test for predicting field susceptibility of rubber clones to CLF will have to be further investigated.

MIP diversity from Trichoderma: Structural considerations and transcriptional modulation during mycoparasitic association with Fusarium solani olive trees

Major intrinsic proteins (MIP) are characterized by a transmembrane pore-type architecture that facilitates transport across biomembranes of water and a variety of low molecular weight solutes. They are found in all parts of life, with remarkable protein diversity. Very little is known about MIP from fungi. And yet, it can legitimately be stated that MIP are pivotal molecular components in the privileged relationships fungi enjoy with plants or soil fauna in various environments. To date, MIP have never been studied in a mycoparasitism situation. In this study, the diversity, expression and functional prediction of MIP from the genus Trichoderma were investigated. Trichoderma spp. genomes have at least seven aquaporin genes. Based on a phylogenetic analysis of the translated sequences, members were assigned to the AQP, AQGP and XIP subfamilies. In in vitro and in planta assays with T. harzianum strain Ths97, expression analyses showed that four genes were constitutively expressed. In a mycoparasitic context with Fusarium solani, the causative agent of fusarium dieback on olive tree roots, these genes were up-regulated. This response is of particular interest in analyzing the MIP promoter cis-regulatory motifs, most of which are involved in various carbon and nitrogen metabolisms. Structural analyses provide new insights into the possible role of structural checkpoints by which these members transport water, H2O2, glycerol and, more generally, linear polyols across the membranes. Taken together, these results provide the first evidence that MIP may play a key role in Trichoderma mycoparasitism lifestyle.