Gene deletion of Corynespora cassiicola cassiicolin Cas1 suppresses virulence in the rubber tree.

Corynespora cassiicola is an ascomycete fungus causing important damages in a wide range of plant hosts, including rubber tree. The small secreted protein cassiicolin is suspected to play a role in the onset of the disease in rubber tree, based on toxicity and gene expression profiles. However, its exact contribution to virulence, compared to other putative effectors, remains unclear. We created a deletion mutant targeting the cassiicolin gene Cas1 from the highly aggressive isolate CCP. Wild-type CCP and mutant ccpΔcas1 did not differ in terms of mycelium growth, sporulation, and germination rate in vitro. Cas1 gene deletion induced a complete loss of virulence on the susceptible clones PB260 and IRCA631, as revealed by inoculation experiments on intact (non-detached) leaves. However, residual symptoms persisted when inoculations were conducted on detached leaves, notably with longer incubation times. Complementation with exogenous cassiicolin restored the mutant capacity to colonize the leaf tissues. We also compared the toxicity of CCP and ccpΔcas1 culture filtrates, through electrolyte leakage measurements on abraded detached leaves, over a range of clones as well as an F1 population derived from the cross between the clones PB260 (susceptible) and RRIM600 (tolerant). On average, filtrate toxicity was lower but not fully suppressed in ccpΔcas1 compared to CCP, with clone-dependent variations. The two QTL, previously found associated with sensitivity to CPP filtrate or to the purified cassiicolin, were no longer detected with the mutant filtrate, while new QTL were revealed. Our results demonstrate that: (1) cassiicolin is a necrotrophic effector conferring virulence to the CCP isolate in susceptible rubber clones and (2) other effectors produced by CCP contribute to residual filtrate toxicity and virulence in senescing/wounded tissues. These other effectors may be involved in saprotrophy rather than necrotrophy.

Endophytes from Wild Rubber Trees as Antagonists of the Pathogen Corynespora cassiicola.

The Corynespora leaf fall disease of rubber trees, caused by the necrotrophic fungus Corynespora cassiicola, is responsible for important yield losses in Asian and African plantations, whereas its impact is negligible in South America. The objective of this study was to identify potential antagonists of C. cassiicola among fungal endophytes (i.e., Pestalotiopsis, Colletotrichum, and Trichoderma spp.) isolated from wild and cultivated rubber trees distributed in the Peruvian Amazon. We first tested the endophytes in dual in vitro confrontation assays against a virulent C. cassiicola isolate (CCP) obtained from diseased rubber trees in the Philippines. All Trichoderma isolates overran the CCP colony, suggesting some antagonistic mechanism, while species from the other genera behaved as mutual antagonists. Trichoderma isolates were then tested through antibiosis assays for their capacity to produce growth-inhibiting molecules. One isolate (LA279), recovered as an endophyte from a wild Hevea guianensis specimen and identified as Trichoderma koningiopsis, showed significant antibiosis capacity. We demonstrated that LA279 was also able to endophytically colonize the cultivated rubber tree species (H. brasiliensis). Under controlled laboratory conditions, rubber plants were inoculated with three Trichoderma strains, including LA279, in combination with the pathogenic CCP. Results showed that 1 week preinoculation with the endophytes differentially reduced CCP mycelial development and symptoms. In conclusion, this study suggests that T. koningiopsis isolate LA279-and derivate compounds-could be a promising candidate for the biological control of the important rubber tree pathogen C. cassiicola.

Transcriptome profiling in susceptible and tolerant rubber tree clones in response to cassiicolin Cas1, a necrotrophic effector from Corynespora cassiicola.

Corynespora cassiicola, a fungal plant pathogen with a large host range, causes important damages in rubber tree (Hevea brasiliensis), in Asia and Africa. A small secreted protein named cassiicolin was previously identified as a necrotrophic effector required for the virulence of C. cassiicola in specific rubber tree clones. The objective of this study was to decipher the cassiicolin-mediated molecular mechanisms involved in this compatible interaction. We comparatively analyzed the RNA-Seq transcriptomic profiles of leaves treated or not with the purified cassiicolin Cas1, in two rubber clones: PB260 (susceptible) and RRIM600 (tolerant). The reads were mapped against a synthetic transcriptome composed of all available transcriptomic references from the two clones. Genes differentially expressed in response to cassiicolin Cas1 were identified, in each clone, at two different time-points. After de novo annotation of the synthetic transcriptome, we analyzed GO enrichment of the differentially expressed genes in order to elucidate the main functional pathways impacted by cassiicolin. Cassiicolin induced qualitatively similar transcriptional modifications in both the susceptible and the tolerant clones, with a strong negative impact on photosynthesis, and the activation of defense responses via redox signaling, production of pathogenesis-related protein, or activation of the secondary metabolism. In the tolerant clone, transcriptional reprogramming occurred earlier but remained moderate. By contrast, the susceptible clone displayed a late but huge transcriptional burst, characterized by massive induction of phosphorylation events and all the features of a hypersensitive response. These results confirm that cassiicolin Cas1 is a necrotrophic effector triggering a hypersensitive response in susceptible rubber clones, in agreement with the necrotrophic-effector-triggered susceptibility model.

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors.

Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species (Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca, and Botrosphaeria dothidea) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector-based classification was found to be highly consistent with the phylogenomic trees. Identification of lineage-specific effectors is a key step toward understanding C. cassiicola virulence and host specialization mechanisms.