Multi-factor regulation of pectate lyase secretion by Colletotrichum gloeosporioides pathogenic on avocado fruits.

Tissue alkalinization during Colletotrichum gloeosporioides attack enhances the expression of PELB, which encodes pectate lyase (PL), and PL secretion, which is considered essential for full virulence. We studied the regulation of PL secretion by manipulation of C. gloeosporioides PELB. PELB was down-regulated by knocking out PAC1, which encodes the PacC transcription factor that regulates gene products with pH-sensitive activities. We functionally characterized a PACC gene homologue, PAC1, from C. gloeosporioides wild-type (WT) Cg-14 and two independent deletion strains, Deltapac1(372)and Deltapac1(761). Loss-of-function PAC1 mutants showed 85% reduction of PELB transcript expression, delayed PL secretion and dramatically reduced virulence, as detected in infection assays with avocado fruits. In contrast, PELB was up-regulated in the presence of carbon sources such as glucose. When glucose was used as a carbon source in the medium for the WT strain and the Deltapac1 mutant at pH 6.0, PELB transcript expression and PL secretion were activated. Other sugars, such as sucrose and fructose (but not galactose), also activated PELB expression. These results suggest that the pH-regulated response is only part of a multi-factor regulation of PELB, and that sugars are also needed to promote the transition from quiescent to active necrotrophic development by the pathogen.

Effect of ammonia production by Colletotrichum gloeosporioides on pelB activation, pectate lyase secretion, and fruit pathogenicity.

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 KNO3 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 KNO3 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.

External pH and nitrogen source affect secretion of pectate lyase by Colletotrichum gloeosporioides.

Accumulation of ammonia and associated tissue alkalinization predispose fruit to attack by Colletotrichum gloeosporioides: 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.