BackgroundThe basidiomycete fungus Moniliophthora perniciosa is the causal agent of Witches' Broom Disease (WBD) in cacao (Theobroma cacao). It is a hemibiotrophic pathogen that colonizes the apoplast of cacao's meristematic tissues as a biotrophic pathogen, switching to a saprotrophic lifestyle during later stages of infection. M. perniciosa, together with the related species M. roreri, are pathogens of aerial parts of the plant, an uncommon characteristic in the order Agaricales. A genome survey (1.9× coverage) of M. perniciosa was analyzed to evaluate the overall gene content of this phytopathogen.ResultsGenes encoding proteins involved in retrotransposition, reactive oxygen species (ROS) resistance, drug efflux transport and cell wall degradation were identified. The great number of genes encoding cytochrome P450 monooxygenases (1.15% of gene models) indicates that M. perniciosa has a great potential for detoxification, production of toxins and hormones; which may confer a high adaptive ability to the fungus. We have also discovered new genes encoding putative secreted polypeptides rich in cysteine, as well as genes related to methylotrophy and plant hormone biosynthesis (gibberellin and auxin). Analysis of gene families indicated that M. perniciosa have similar amounts of carboxylesterases and repertoires of plant cell wall degrading enzymes as other hemibiotrophic fungi. In addition, an approach for normalization of gene family data using incomplete genome data was developed and applied in M. perniciosa genome survey.ConclusionThis genome survey gives an overview of the M. perniciosa genome, and reveals that a significant portion is involved in stress adaptation and plant necrosis, two necessary characteristics for a hemibiotrophic fungus to fulfill its infection cycle. Our analysis provides new evidence revealing potential adaptive traits that may play major roles in the mechanisms of pathogenicity in the M. perniciosa/cacao pathosystem.
1 The effect of purified crotapotin, a non-toxic non-enzymatic chaperon protein normally complexed to a phospholipase A2 (PLA2) in South America rattlesnake venom, was studied in the acute inflammatory response induced by carrageenin (1 mg/paw), compound 48/80 (3 jig/paw) and 5-hydroxytryptamine (5-HT) (3 jig/paw) in the rat hind-paw. The effects of crotapotin on platelet aggregation, mast cell degranulation and eicosanoid release from guinea-pig isolated lung were also investigated.2 Subplantar co-injection of crotapotin (1 and 10 jig/paw) with carrageenin or injection of crotapotin (10 jig/paw) into the contralateral paw significantly inhibited the carrageenin-induced oedema. This inhibition was also observed when crotapotin (10-30 jig/paw) was administered either intraperitoneally or orally. Subplantar injection of heated crotapotin (15 min at 60°C) failed to inhibit carrageenininduced oedema. Subplantar injection of crotapotin (10 jig/paw) also significantly inhibited the rat paw oedema induced by compound 48/80, but it did not affect 5-HT-induced oedema. 3 In adrenalectomized animals, subplantar injection of crotapotin markedly inhibited the oedema induced by carrageenin. The inhibitory effect of crotapotin was also observed in rats depleted of histamine and 5-HT stores.4 Crotapotin (30 jig/paw) had no effect on either the histamine release induced by compound 48/80 in vitro or on the platelet aggregation induced by both arachidonic acid (1 mM) and platelet activating factor (1 jiM) in human platelet-rich plasma. The platelet aggregation and thromboxane B2 (TXB2) release induced by thrombin (100 mu ml-') in washed human platelets were also not affected by crotapotin. In addition, crotapotin (10 jig/paw) did not affect the release of 6-oxo-prostaglandin Flc, and TXB2 induced by ovalbumin in sensitized guinea-pig isolated lungs. 5 Our results indicate that the anti-inflammatory activity of crotapotin is not due to endogenous corticosteroid release or inhibition of cyclo-oxygenase activity. It is possible that crotapotin may interact with extracellular PLA2 generated during the inflammatory process thereby reducing its hydrolytic activity.
The complete amino acid sequence of bothropstoxin-I (BthTX-I), a myotoxin isolated from Bothrops jararacussu snake venom, is reported. The results show that BthTX-I is a Lys49 phospholipase A2 (PLA2)-like protein composed of a single polypeptide chain of 121 amino acid residues (M(r) = 13,720), containing one methionine and 14 half-cystines. Although deprived of any detectable PLA2 activity, BthTX-I reveals a high degree of sequence homology with Asp49-PLA2s and with other Lys49-myotoxins. Critical mutations--such as Leu5 for Phe5; Gln11 for X11; Asn28 for Tyr28; Leu32 for Gly32; Lys49 for Asp49; and Asp71 for Asn71--which are apparently involved with the decreasing or elimination of PLA2 activity, have been detected. The same mutations occurred in myotoxin II from Bothrops asper venom, but five extra changes--namely, Pro90 for Ser90; Gly111 for Asn111; His120 for Tyr120; Phe124 for Leu124; and Pro132 for Ala132--have been found relative to myotoxin II.
Moniliophthora perniciosa is a hemibiotrophic fungus that causes witches' broom disease (WBD) in cacao. Marked dimorphism characterizes this fungus, showing a monokaryotic or biotrophic phase that causes disease symptoms and a later dikaryotic or saprotrophic phase. A combined strategy of DNA microarray, expressed sequence tag, and real-time reverse-transcriptase polymerase chain reaction analyses was employed to analyze differences between these two fungal stages in vitro. In all, 1,131 putative genes were hybridized with cDNA from different phases, resulting in 189 differentially expressed genes, and 4,595 reads were clusterized, producing 1,534 unigenes. The analysis of these genes, which represent approximately 21% of the total genes, indicates that the biotrophic-like phase undergoes carbon and nitrogen catabolite repression that correlates to the expression of phytopathogenicity genes. Moreover, downregulation of mitochondrial oxidative phosphorylation and the presence of a putative ngr1 of Saccharomyces cerevisiae could help explain its lower growth rate. In contrast, the saprotrophic mycelium expresses genes related to the metabolism of hexoses, ammonia, and oxidative phosphorylation, which could explain its faster growth. Antifungal toxins were upregulated and could prevent the colonization by competing fungi. This work significantly contributes to our understanding of the molecular mechanisms of WBD and, to our knowledge, is the first to analyze differential gene expression of the different phases of a hemibiotrophic fungus.
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