Moniliophthora perniciosa development: key genes involved in stress-mediated cell wall organization and autophagy.

Moniliophthora perniciosa is a basidiomycete responsible for the witches' broom disease in cacao (Theobroma cacao L.). Chitin synthase (CHS), chitinase (CHIT) and autophagy (ATG) genes have been associated to stress response preceding the formation of basidiocarp. An analysis of literature mining, interactomics and gene expression was developed to identify the main proteins related to development, cell wall organization and autophagy in M. perniciosa. TORC2 complex elements were identified and were involved in the response to the nutrient starvation during the fungus development stages preceding the basidiocarp formation. This complex interacted with target proteins related to cell wall synthesis and to polarization and cell division (FKS1, CHS, CDC42, ROM2). Autolysis and autophagy processes were associated to CHIT2, ATG8 and to the TORC1 complex (TOR1 and KOG1), which is central in the upstream signalization of the stress response due to nutrient starvation and growth regulation. Other important elements that participate to steps preceding basidiocarp formation were also identified (KOG1, SSZ1, GDI1, FKS1, CCD10, CKS1, CDC42, RHO1, AVO1, BAG7). Similar gene expression patterns during fungus reproductive structure formation and when treated by rapamycin (a nutritional related-autophagy stress agent) were observed: cell division related-genes were repressed while those related to autolysis/autophagy were overexpressed.

Mycelial development preceding basidioma formation in Moniliophthora perniciosa is associated to chitin, sugar and nutrient metabolism alterations involving autophagy.

We identified and characterized two chitinases, named MpCHIT1 and MpCHIT2, from the fungus Moniliophthora perniciosa - the etiologic agent of witches' broom disease in cacao tree (Theobroma cacao L.) - during its development, mainly in the mycelia phases preceding the basidioma formation. The expression of MpCHIT1 and MpCHIT2, together with MpCHS and MpATG8 (chitin synthase and autophagy genes, respectively), was analyzed during the M. perniciosa growth and development on bran-based solid medium as well as in liquid medium containing H2O2 or rapamycin (oxidative and nutritional related-autophagy stress agents, respectively). In order to link the expression of chitin metabolism-related genes to nutritional composition influencing fungus development, we also quantified total and reduced sugars, as well as macro- and micronutrients in the bran-based solid medium. The expression analysis showed that the MpCHS expression increased through mycelial development and then decreased in the primordium and basidioma phases, while the expression of MpCHIT1 and MpCHIT2 was higher in basidioma and primordium phases, respectively. Moreover, the expression pattern of MpCHIT1 and MpCHIT2 is distinct, the second correlated with the MpATG8 expression pattern and possibly with autophagy process, while the first may be related to the basidioma formation. The quantification of total and reduced sugars, as well as macro- and micronutrients supported the idea that the cell wall restructuration due to MpCHS, MpCHIT1 and MpCHIT2 is related to stress and fungal nutrient reallocation, allowing the formation and development of the basidioma. Experiments involving M. perniciosa growth on liquid medium containing H2O2 or rapamycin showed that MpCHIT1 and MpCHIT2 were over-expressed in response to oxidative but also to nutritional related-autophagy stresses. Interestingly, the expression level of MpCHS, MpCHIT1 and MpCHIT2 in presence of rapamycin is similar to the one observed in the primordium and basidioma from bran-based solid medium. The analysis of the overall data allowed designing a general scheme of chitin metabolism and autophagy during M. perniciosa development, focusing on the mycelium phases as crucial and environmentally influenced steps preceding the primordium and basidioma formation. These data support the idea that the nutritional environment of M. perniciosa influences its development and life cycle.

Protein extraction for proteome analysis from cacao leaves and meristems, organs infected by Moniliophthora perniciosa, the causal agent of the witches' broom disease.

Preparation of high-quality proteins from cacao vegetative organs is difficult due to very high endogenous levels of polysaccharides and polyphenols. In order to establish a routine procedure for the application of proteomic and biochemical analysis to cacao tissues, three new protocols were developed; one for apoplastic washing fluid (AWF) extraction, and two for protein extraction--under denaturing and nondenaturing conditions. The first described method allows a quick and easy collection of AWF--using infiltration-centrifugation procedure--that is representative of its composition in intact leaves according to the smaller symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the nondenaturing protein extraction and subsequent enzymatic activity detection. It is expected that the protocols described here could help to develop high-level proteomic and biochemical studies in cacao also being applicable to other recalcitrant plant tissues.

Use of response surface methodology to examine chitinase regulation in the basidiomycete Moniliophthora perniciosa.

We report here the first analysis of chitinase regulation in Moniliophthora perniciosa, the causal agent of the witches' broom disease of cacao. A multivariate statistical approach was employed to evaluate the effect of several variables, including carbon and nitrogen sources and cultivation time, on M. perniciosa non-secreted (detected in mycelium, i.e. in symplasm and cell wall) and secreted (detected in the culture medium) chitinase activities. Non-secreted chitinase activity was enhanced by peptone and chitin and repressed by glucose. Chitinase secretion was increased by yeast extract alone or in combination with other nitrogen sources, and by N-acetylglucosamine, and repressed in presence of chitin. The best cultivation times for non-secreted and secreted chitinase activities were 30 and 20 d, respectively. However, chitinase activity was always higher in the mycelium than in the culture medium, suggesting a relatively poor chitinase secretion activity. Conversely, higher mycelial growth was observed when the activity of the non-secreted chitinase was at its lowest, i.e. when the fungus was grown on glucose and yeast extract as sources of carbon and nitrogen, respectively. Conversely, the induction of non-secreted chitinase activity by chitin decreased the mycelium growth. These results suggest that the culture medium, by the induction or repression of chitinases, affected the hyphal growth. Thus, as an essential component of M. perniciosa growth, chitinases may be a potential target for strategies to control disease.