Accumulation of ammonia and associated tissue alkalinization predispose fruit to attack by Colletotrichumgloeosporioides. As the external pH increases from 4.0 to 6.0, pectate lyase (PL) and other extracellular proteins are secreted and accumulate. At pH 4.0 neither pelB (encoding PL) transcription nor PL secretion were detected; however, they were detected as the pH increased. Nitrogen assimilation also was required for PL secretion at pH 6.0. Both inorganic and organic nitrogen sources enhanced PL secretion at pH 6.0, but neither was sufficient for PL secretion at pH 4.0. Sequence analysis of the 5 upstream region of the pelB promoter revealed nine putative consensus binding sites for the Aspergillus transcription factor PacC. Consistent with this result, the transcript levels of pac1 (the C. gloeosporioides pacC homologue) and pelB increased in parallel as a function of pH. Our results suggest that the ambient pH and the nitrogen source are independent regulatory factors for processes linked to PL secretion and virulence of C. gloeosporioides.Colletotrichum gloeosporioides is a filamentous ascomycete phytopathogen that attacks the fruits of many hosts before and after harvest. During colonization by the pathogen ammonia accumulates in the host (14), which creates the alkaline pH environment necessary for the activities of some extracellular lyase enzymes, including pectate lyases (PLs) (3,4,26,27). PL has been implicated as a virulence factor of C. gloeosporioides in avocado fruit (29), and its expression is strongly affected by alkalinization. Thus, environmental conditions that affect PL secretion may be important virulence factors for C. gloeosporioides.The alkalinization of the tissue and the increased virulence of C. gloeosporioides also may depend on the availability of exogenous nitrogen, which can be converted to NH 4 ϩ . The increase in the amount of NH 4 ϩ thus increases the external pH, which leads to expression of different genes, including pelB, the gene encoding PL, and other genes encoding secreted pectolytic enzymes. In Aspergillus nidulans and Sclerotinia sclerotiorum an ambient-pH-sensing signal transduction pathway affects expression of genes encoding several secreted and outer-membrane-bound proteins, as well as enzymes that synthesize exportable metabolites (2,5,7,8,11,17,19,24). The product of the pacC gene is the terminal component of the pH signaling pathway and the transcription regulator of pH-dependent gene expression (2). This protein has a zinc finger DNA-binding domain with the core DNA consensus binding site 5Ј-GCCARG-3Ј (24).In this study our main objective was to determine the importance of the nitrogen source and the external pH in secretion of the virulence factor PL with respect to the ambient pH transcriptional regulator pacC. We hypothesized that nitrogen source availability and the ambient pH are two independent signals for transcriptional regulation of genes required for the disease processes of C. gloeosporioides and possibly other pathogens. MATERIALS AND METHODSStrai...
The accumulation of ammonia and associated tissue alkalinization predispose avocado fruit to attack by Colletotrichum gloeosporioides. Secretion of ammonia by C. gloeosporioides in the presence of KNO 3 was induced by decreasing the pH from 7.0 to 4.0. When the fungus was grown at pH 4.0 or 6.0 in the absence of a nitrogen source, ammonia did not accumulate, and neither pelB (encoding pectate lyase) transcription nor pectate lyase secretion was detected. Under these nitrogen starvation conditions, only transcriptional activation of areA, which encodes the global nitrogen regulator, was detected. pelB transcription and pectate lyase secretion were both detected when C. gloeosporioides was grown at pH 6.0 in the presence of ammonia accumulated from different nitrogen sources. The early accumulation of ammonia induced early pelB expression and pectate lyase secretion. As the external pH increased from 4.0 to 6.0, transcripts of pac1, the C. gloeosporioides pacC homolog, also could be detected. Nit mutants of C. gloeosporioides, which cannot utilize KNO 3 as a nitrogen source, did not secrete ammonia, alkalinize the medium, or secrete pectate lyase. If Nit mutants were grown at pH 6.0 in the presence of glutamate, then pectate lyase secretion was induced. Infiltration of 0.1 M ammonium hydroxide at pH 10 into ripening avocado fruits enhanced the activation of quiescent infection and symptom development by C. gloeosporioides. These results suggest that ambient pH alkalinization resulting from ammonia accumulation and the availability of ammonia as a nitrogen source independently regulate pelB expression, pectate lyase secretion, and virulence of C. gloeosporioides. These data suggest that alkalinization during C. gloeosporioides infection is important for its transformation from the quiescent biotrophic stage to the necrotrophic stage of fungal colonization in the fruit host.Colletotrichum gloeosporioides is a filamentous ascomycete plant pathogen that attacks the fruits of a variety of pre-and postharvest hosts. C. gloeosporioides germinates on avocado (Persea americana Miller var. drymifolia [Schlechtendal and Chamisso] S. F. Blake) fruit peel and forms appressoria. Infection hyphae from the appressoria penetrate the epidermal cells of the avocado exocarp but remain quiescent until the fruit ripens. The resistance of unripe fruit to fungal attack during quiescence is related to the presence of preformed or inducible antifungal compounds and the lack of secretion of fungal pathogenicity factors. The activation of quiescent infection results from a decline in the concentration of preformed antifungal compounds followed by the activation of fungal pathogenicity factors (27, 28). Pectate lyase (PL) has been implicated as a virulence factor of C. gloeosporioides in avocado fruit (26), and its expression is strongly affected by alkalinization (8). Alkalinization of the tissue occurs naturally during fruit ripening, where the pH of the pericarp increases from 5.2 to 6.1. However, the pathogen also helps increase the amoun...
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