High-resolution transcript profiling of the atypical biotrophic interaction between Theobroma cacao and the fungal pathogen Moniliophthora perniciosa.

Witches' broom disease (WBD), caused by the hemibiotrophic fungus Moniliophthora perniciosa, is one of the most devastating diseases of Theobroma cacao, the chocolate tree. In contrast to other hemibiotrophic interactions, the WBD biotrophic stage lasts for months and is responsible for the most distinctive symptoms of the disease, which comprise drastic morphological changes in the infected shoots. Here, we used the dual RNA-seq approach to simultaneously assess the transcriptomes of cacao and M. perniciosa during their peculiar biotrophic interaction. Infection with M. perniciosa triggers massive metabolic reprogramming in the diseased tissues. Although apparently vigorous, the infected shoots are energetically expensive structures characterized by the induction of ineffective defense responses and by a clear carbon deprivation signature. Remarkably, the infection culminates in the establishment of a senescence process in the host, which signals the end of the WBD biotrophic stage. We analyzed the pathogen's transcriptome in unprecedented detail and thereby characterized the fungal nutritional and infection strategies during WBD and identified putative virulence effectors. Interestingly, M. perniciosa biotrophic mycelia develop as long-term parasites that orchestrate changes in plant metabolism to increase the availability of soluble nutrients before plant death. Collectively, our results provide unique insight into an intriguing tropical disease and advance our understanding of the development of (hemi)biotrophic plant-pathogen interactions.

Suppression of Plant Immunity by Fungal Chitinase-like Effectors.

Crop diseases caused by fungi constitute one of the most important problems in agriculture, posing a serious threat to food security [1]. To establish infection, phytopathogens interfere with plant immune responses [2, 3]. However, strategies to promote virulence employed by fungal pathogens, especially non-model organisms, remain elusive [4], mainly because fungi are more complex and difficult to study when compared to the better-characterized bacterial pathogens. Equally incomplete is our understanding of the birth of microbial virulence effectors. Here, we show that the cacao pathogen Moniliophthora perniciosa evolved an enzymatically inactive chitinase (MpChi) that functions as a putative pathogenicity factor. MpChi is among the most highly expressed fungal genes during the biotrophic interaction with cacao and encodes a chitinase with mutations that abolish its enzymatic activity. Despite the lack of chitinolytic activity, MpChi retains substrate binding specificity and prevents chitin-triggered immunity by sequestering immunogenic chitin fragments. Remarkably, its sister species M. roreri encodes a second non-orthologous catalytically impaired chitinase with equivalent function. Thus, a class of conserved enzymes independently evolved as putative virulence factors in these fungi. In addition to unveiling a strategy of host immune suppression by fungal pathogens, our results demonstrate that the neofunctionalization of enzymes may be an evolutionary pathway for the rise of new virulence factors in fungi. We anticipate that analogous strategies are likely employed by other pathogens.

Novel receptor-like kinases in cacao contain PR-1 extracellular domains.

Members of the pathogenesis-related protein 1 (PR-1) family are well-known markers of plant defence responses, forming part of the arsenal of the secreted proteins produced on pathogen recognition. Here, we report the identification of two cacao (Theobroma cacao L.) PR-1s that are fused to transmembrane regions and serine/threonine kinase domains, in a manner characteristic of receptor-like kinases (RLKs). These proteins (TcPR-1f and TcPR-1g) were named PR-1 receptor kinases (PR-1RKs). Phylogenetic analysis of RLKs and PR-1 proteins from cacao indicated that PR-1RKs originated from a fusion between sequences encoding PR-1 and the kinase domain of a LecRLK (Lectin Receptor-Like Kinase). Retrotransposition marks surround TcPR-1f, suggesting that retrotransposition was involved in the origin of PR-1RKs. Genes with a similar domain architecture to cacao PR-1RKs were found in rice (Oryza sativa), barrel medic (Medicago truncatula) and a nonphototrophic bacterium (Herpetosiphon aurantiacus). However, their kinase domains differed from those found in LecRLKs, indicating the occurrence of convergent evolution. TcPR-1g expression was up-regulated in the biotrophic stage of witches' broom disease, suggesting a role for PR-1RKs during cacao defence responses. We hypothesize that PR-1RKs transduce a defence signal by interacting with a PR-1 ligand.