Carbon regulation of environmental pH by secreted small molecules that modulate pathogenicity in phytopathogenic fungi

Abstract

Fruit pathogens can contribute to the acidification or alkalinization of the host environment. This capability has been used to divide fungal pathogens into acidifying and/or alkalinizing classes. Here, we show that diverse classes of fungal pathogens-Colletotrichum gloeosporioides, Penicillium expansum, Aspergillus nidulans and Fusarium oxysporum-secrete small pH-affecting molecules. These molecules modify the environmental pH, which dictates acidic or alkaline colonizing strategies, and induce the expression of PACC-dependent genes. We show that, in many organisms, acidification is induced under carbon excess, i.e. 175 mm sucrose (the most abundant sugar in fruits). In contrast, alkalinization occurs under conditions of carbon deprivation, i.e. less than 15 mm sucrose. The carbon source is metabolized by glucose oxidase (gox2) to gluconic acid, contributing to medium acidification, whereas catalysed deamination of non-preferred carbon sources, such as the amino acid glutamate, by glutamate dehydrogenase 2 (gdh2), results in the secretion of ammonia. Functional analyses of Δgdh2 mutants showed reduced alkalinization and pathogenicity during growth under carbon deprivation, but not in high-carbon medium or on fruit rich in sugar, whereas analysis of Δgox2 mutants showed reduced acidification and pathogencity under conditions of excess carbon. The induction pattern of gdh2 was negatively correlated with the expression of the zinc finger global carbon catabolite repressor creA. The present results indicate that differential pH modulation by fruit fungal pathogens is a host-dependent mechanism, affected by host sugar content, that modulates environmental pH to enhance fruit colonization.

Keywords: carbon regulation of pathogenicity; pH regulation; pathogenicity.

Figure 1

Effects of carbon concentration on the induction of medium alkalinization or acidification by Colletotrichum gloeosporioides and the induction of PACC‐regulated acid‐ and alkaline‐expressed genes. Fungal mycelia of C. gloeosporioides (Cg) were grown in primary rich medium for 3 days and then transferred to secondary medium containing sucrose at 15 mm (■) or 175 mm (□), adjusted to pH 5, for 72 h. The effect of sucrose concentration was evaluated on the modulation of (A) pH, (B) gluconic acid (GLA) accumulation, (C) ammonia accumulation and (D) pacC relative expression (RE). RNA for pacC RE was extracted from mycelia at the indicated time points after transfer to inducing medium, and cDNA was used for quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). RNA for the evaluation of pacC‐regulated genes was extracted from mycelia 24 h after transfer to inducing medium. (E) The acid‐expressed genes encoded glucanase b (Glub), aspartic type (Asp), proline oxidase (PO), flavin reductase fmn‐binding protein (FRFBP), Tim barrel metal‐dependent hydrolase (TBMDH), acetamidase formamidase (AF), mannan endo‐β‐mannosidase (MEBM), amino acid permease family (AAP), mfs allantotate (MAT), endopolygalacturonase (EPG) and β‐endoglucanase (β‐EGlu). (f) The PACC alkaline‐induced genes encoded polysaccharide deacetylase family protein (PDA), sugar transport protein (STP), abc transporter (AT), cellulose synthase catalytic subunit (CSCS), general amino acid permease (GAAP), calcium‐transporting ATPase 3 (CTA3), pectate lyase b (PelB), histidine acid phosphatase (HAP) and high‐affinity glucose transporter (Ght2). The 18S gene was used to normalize the expression data for each fungus. Values represent means ± standard error (SE) of four biological replicates. The experiments were repeated three times. PI, post‐inoculation.

Figure 2

Relative expression (RE) of gdh2 (A), gox2 (B) and pacC (C) in Colletotrichum gloeosporioides grown in different carbon levels. Fungal mycelia were grown in primary rich medium for 3 days and then transferred to secondary media (SM) medium containing sucrose at 15 mm (■) or 175 mm (□), adjusted to pH 5. RNA was extracted from mycelia at the indicated time points after transfer to secondary medium, and cDNA was used for quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). The 18S gene was used to normalize the expression data. Values represent means ± standard error (SE) of duplicates. Experiments were repeated three times. PI, post‐inoculation.

Figure 3

Effects of carbon levels on alkalinization or acidification of the medium by wild‐type (WT) and mutant strains of Colletotrichum gloeosporioides. (A, B) Δgdh2. (C, D) Δgox2. (A, C) Ammonia and (B, D) gluconic acid (GLA) accumulation. Fungal mycelia were grown in primary rich medium (M₃S) for 3 days and then transferred to secondary medium (SM) containing sucrose at 15 mm (■▲) or 175 mm (□Δ), adjusted to pH 5. Values represent means ± standard error (SE) of four biological replicates. Experiments were repeated three times. PI, post‐inoculation.

Figure 4

Effect of carbon levels on the induction of medium alkalinization or acidification by Penicillium expansum (Pe), Aspergillus nidulans (An) and Fusarium oxysporum (Fo), and relative expression (RE) of pacC. (A) Fungal mycelia were grown in primary rich medium for 2–3 days and then transferred to secondary medium containing sucrose at 15 mm (■) or 175 mm (□), adjusted to pH 5, for 72 h. The effect of sucrose concentration was evaluated on pH modulation, and ammonia and gluconic acid (GLA) accumulation, for all three fungal species. Values represent means ± standard error (SE) of quadruplets. Experiments were repeated three times. (B, C) RE of pacC in Pe and Fo, respectively, during induction of alkalinization or acidification of the growth medium. RNA was extracted from mycelia at the indicated time points after transfer to inducing medium, and cDNA was used for quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Species‐specific housekeeping genes (Table S3) were used to normalize the expression data for each fungus. Values represent means ± SE of duplicates. Experiments were repeated three times. PI, post‐inoculation.

Figure 5

Effects of carbon levels on the induction of medium alkalinization or acidification by Colletotrichum gloeosporioides (A) and Penicillium expansum (B). Fungal mycelia were grown in primary rich medium for 2–3 days and then transferred to secondary medium adjusted to pH 5 and containing 3 g/L tryptone amended with: 15 (■), 25 (▲), 50 (•), 100 (Δ) or 175 mm (□) sucrose. Values represent means ± standard error (SE) of quadruplets. Experiments were repeated three times. PI, post‐inoculation.

Figure 6

Effects of carbon levels on relative expression (RE) of creA, gox2 and gdh2 in Colletotrichum gloeosporioides (A–C) and Penicillium expansum (D–F). Fungal mycelia were grown in primary rich medium for 3 days and then transferred to secondary medium containing increasing concentrations (15–175 mm) of sucrose and 3 g/L tryptone, adjusted to pH 5. RNA was extracted from mycelia at 48 h after transfer to inducing medium and cDNA was used for quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) of creA. Species‐specific housekeeping genes (Table S3) were used to normalize the expression data for each fungus. Values represent means ± standard error (SE) of duplicates. Experiments were repeated three times.

Figure 7

Differential modulation of pH by Colletotrichum gloeosporioides on different fruits and effect on PACC‐regulated genes. (A–C) avocado; (D–F) tomato; (G–I) plum; (J) PACC differentially regulated genes. Fruit pericarps were wound inoculated with a 10 µL suspension containing 10⁶ spores/mL, and infected fruits were incubated at 25 °C, ∼85% relative humidity (RH), for 3 days. Healthy and decayed tissues were sampled and used for the determination of pH, ammonia and gluconic acid (GLA). Values represent means ± standard error (SE) of four or five inoculations, each of eight fruits (c. 32 replications). Three days after fruit inoculation, RNA was extracted from the infected fruit tissues and the cDNA was used for quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). The 18S gene was used to normalize the expression data. Experiments were repeated three times. *P < 0.05 according to Student's t‐test. PI, post‐inoculation.

Figure 8

Differential modulation of pathogencity and pH by Colletotrichum gloeosporioides wild‐type (WT), Δgdh2 and Δgox2 strains on tomato (A–D, I) and plum (E–H). Fruits were wound inoculated with 10‐µL suspensions of WT, Δgdh2 and Δgox2 strains containing 10⁶ spores/mL. Infected fruits were incubated at 25 °C, ∼85% relative humidity (RH), for 3 days. Inoculation of sucrose‐treated tomato fruits with Δgox2 was carried out as described in Experimental procedures. The decay was examined after 3 days, and healthy and decayed tissues were sampled and subjected to pH analysis. Values represent means ± standard error (SE) of four or five inoculations, each of seven or eight fruits. The experiments were repeated three times. DDW, double distilled water; PI, post‐inoculation.

Figure 9

pH modulation in plum and tomato by Colletotrichum gloeosporioides and Penicillium expansum. Fruits were wound inoculated with 10‐µL suspensions containing 10⁶ spores/mL, and infected fruits were incubated at 25 °C, ∼85% relative humidity (RH), for 3 days. Healthy and decayed tissues were sampled and used for pH determination. Values represent means ± standard error (SE) of five inoculations, each of seven or eight fruits. The experiments were repeated three times.