Host-tissue alkalinization via ammonia accumulation is key to Colletotrichum spp. colonization. Using macroarrays carrying C. gloeosporioides cDNAs, we monitored gene expression during the alkalinization process. A set of genes involved in synthesis and catabolism of ammonia accumulation were identified. Expression of NAD(+)-specific glutamate dehydrogenase (GDH2, encoding ammonia synthesis) and the ammonia exporter AMET were induced at pH 4.0 to 4.5. Conversely, ammonia uptake and transcript activation of the ammonia and glutamate importers (MEP and GLT, respectively) and glutamine synthase (GS1) were higher at pH 6.0 to 7.0. Accumulated ammonia in the wild-type mycelium decreased during ambient alkalinization, concurrent with increased GS1 expression. Deltapac1 mutants of C. gloeosporioides, which are sensitive to alkaline pH changes, showed upregulation of the acid-expressed GDH2 and downregulation of the alkaline-expressed GS1, resulting in 60% higher ammonia accumulation inside the mycelium. Deltagdh2 strains of C. gloeosporioides, impaired in ammonia production, showed 85% inhibition in appressorium formation followed by reduced colonization on avocado fruit (Persea americana cv. Fuerte) pericarp, while exogenic ammonia addition restored appressoria formation. Thus the modulation of genes involved in ammonia metabolism and catabolism by C. gloeosporioides is ambient pH-dependent. Aside from its contribution to necrotrophic stages, ammonia accumulation by germinating spores regulates appressorium formation and determines the initiation of biotrophic stages of avocado-fruit colonization by Colletotrichum spp.

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.

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