Transcriptomic Analysis of Trichoderma atroviride Overgrowing Plant-Wilting Verticillium dahliae Reveals the Role of a New M14 Metallocarboxypeptidase CPA1 in Biocontrol

Abstract

Verticillium dahliae, a vascular-colonizing fungus, causes economically important wilt diseases in many crops, including olive trees. Trichoderma spp. have demonstrated an effective contribution as biocontrol agents against this pathogen through a variety of mechanisms that may involve direct mycoparasitism and antibiosis. However, molecular aspects underlaying Trichoderma-V. dahliae interactions are not well known yet due to the few studies in which this pathogen has been used as a target for Trichoderma. In the present study, Trichoderma atroviride T11 overgrew colonies of V. dahliae on agar plates and inhibited growth of highly virulent defoliating (D) V. dahliae V-138I through diffusible molecules and volatile organic compounds produced before contact. A Trichoderma microarray approach of T11 growing alone (CON), and before contact (NV) or overgrowing (OV) colonies of V-138I, helped to identify 143 genes that differed significantly in their expression level by more than twofold between OV and CON or NV. Functional annotation of these genes indicated a marked up-regulation of hydrolytic, catalytic and transporter activities, and secondary metabolic processes when T11 overgrew V-138I. This transcriptomic analysis identified peptidases as enzymatic activity overrepresented in the OV condition, and the cpa1 gene encoding a putative carboxypeptidase (ID number 301733) was selected to validate this study. The role of cpa1 in strain T11 on antagonism of V-138I was analyzed by a cpa1-overexpression approach. The increased levels of cpa1 expression and protease activity in the cpa1-overexpressed transformants compared to those in wild-type or transformation control strains were followed by significantly higher antifungal activity against V-138I in in vitro assays. The use of Trichoderma spp. for the integrated management of plant diseases caused by V. dahliae requires a better understanding of the molecular mechanisms underlying this interaction that might provide an increase on its efficiency.

Keywords: Verticillium wilt; carboxypeptidase; cpa1-overexpressed mutants; microarray; mycoparasitism; secondary metabolism.

FIGURE 1

Microarrays data analysis and experimental setup of Trichoderma atroviride T11 against Verticillium dahliae V-138I. (A) Venn diagram of differentially expressed genes within the set of microarray data. Gene significance was assigned to more than twofold change (FDR: 0.15). Comparative analysis is showed for T11 overgrowing V-138I (OV) in comparison with those in T11 grown at 5 mm from V-138I (NV) and in T11 grown alone (CON). Total number of genes and their subgroups of up- and down-regulated genes are showed for each comparative analysis. The intersection between circles displays the total number of genes up- and down-regulated differently expressed under the overgrowing condition (OV). (B) Scheme of the experimental growth conditions designed for the microarrays. T. atroviride T11 and V. dahliae V-138I, that was inoculated 4 days in advance, were grown onto cellophane-covered PDA plates until samples were collected. T11 is drawn in dark gray color and V-138I is delineated in light gray color. Black oval dots represent the sampling areas that were collected for RNA extraction. CON, T11 grown alone; NV, T11 grown at 5 mm from V-138I; OV, T11 overgrowing V-138I; NT, T11 confronted with itself for RTqPCR validation. ^∗5-mm space left between both organisms.

FIGURE 2

RTqPCR expression analysis of ten JGI-referred genes of set of 143 genes differently expressed when T. atroviride T11 overgrows V. dahliae V-138I (OV). Expression levels were obtained by comparing the expression of these genes in T11 grown at 5 mm from V-138I (NV), T11 grown at 5 mm from itself (NT) and T11 grown alone (CON) conditions. Identification in the T. atroviride genome (JGI) is as follows: exo-β-1,3-glucanase (48371), chitinase (52592), GTP-binding protein (224184), oligopeptide transporter (232557), acyl-CoA thioesterase 2 (282317), metalloprotease (179435), cytochrome P450 monooxygenase (295844), alkaline proteinase (302419), carboxypeptidase A1 (301733), and FAD-monooxygenase (32449). Ct values were referred to the CON condition as a basal reference. Data are the mean of three biological replicates and are displayed as the log10 of the relative quantity (RQ, 2^-ΔΔCt) of target genes compared with the quantity of actin gene used as a reference.

FIGURE 3

Trichoderma atroviride T11 and cpa1-overexpressed strains characterization. (A) Relative expression of cpa1 gene transcripts compared with the quantity of the reference gene (actin). Ct values were referred to MM supplemented with 2% glucose as a basal reference. Data are the mean of three biological replicates and are displayed as the change fold differences (RQ, 2^-ΔΔCt). (B) Protease activity measured with 10 μl of protein extracts. Total activity corresponded to mmol of azocasein hydrolyzed in 1 min, and specific activity corresponded to mmol of azocasein hydrolyzed in 1 min per mg of protein. Tests were performed in triplicate and using, at least, three biological replicates, and the data represent mean values with standard deviations. (C,D) Antifungal activity measured after 72 h with volumes of 10, 25, and 50 μl of protein extracts unboiled (C) and boiled (D). Protein extracts were obtained in all cases from supernatants collected after growing Trichoderma strains in MM supplemented with 2% glucose for 24 h. Values with different superscript letters are significantly different according to Tukey’s test (P < 0.01).