External pH and Nitrogen Source Affect Secretion of Pectate Lyase by Colletotrichum gloeosporioides

Abstract

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...

Extracellular enzymes ofColletotrichum fructicola isolates associated to Apple bitter rot and Glomerella leaf spot

“…The Colletotrichum ability to change the environmental pH from acidic to neutral has been known for decades (Kubicek et al 2014). This species produces ammonia in culture medium and in infected tissues causing alkalisation of the medium and consequently favours enzymatic secretion (Prusky et al 2001; Drori et al 2003). Thus, secretion of some extracellular enzymes, i.e.…”

“…pnlA, pelB are also secreted only at pHs higher than 6.0 (Yakoby et al 2000, Drori et al 2003; Prusky et al 2013). Besides the pH, efficacy of pectolytic enzymes to degrade cell walls can also depend upon the structure of host cell (Kubicek et al 2014) and the different isoforms of PNLs and PGs during colonisation of infected tissues (Shih et al 2000; Yakoby et al 2001).…”

“…On the other hand, PNL is important especially during necrotrophic phase, but is also involved in the switch from biotrophic to necrotrophic phase (Shih et al 2000). Specific conditions in the host tissue, such as, temperature and pH, are also important factors for regulation of gene expression (Prusky et al 2001; Drori et al 2003) and subsequent enzyme production (Yakoby et al 2001). …”

Cloning and characterization of an endo--1,4-xylanase gene from Colletotrichum lindemuthianum and phylogenetic analysis of similar genes from phytopathogenic fungus

“…A number of evidences indicate a role of ambient pH in the regulation of production of pectinolytic enzymes in fungi such as Penicillium paxilli (Szajer and Czajer, 1985), P. itallicum (Alaña et al, 1989), the avocadopathogen C. gloeosporioides (Drori et al, 2003;KramerHaimovich et al, 2006;Yakoby et al, 2000), Trametes trogii (Levin and Forchiassin, 1998) and Aspergillus oryzae (Fontana and Silveira, 2012). In the latter, alkalinization during fruit infection is necessary for the conversion of the biotrophic stage into the necrotrophic stage (Kramer-Haimovich et al, 2006).…”

Effect of Ammonia Production by Colletotrichum gloeosporioides on pelB Activation, Pectate Lyase Secretion, and Fruit Pathogenicity

“…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.…”