The putative protein methyltransferase LAE1 of Trichoderma atroviride is a key regulator of asexual development and mycoparasitism

Abstract

In Ascomycota the protein methyltransferase LaeA is a global regulator that affects the expression of secondary metabolite gene clusters, and controls sexual and asexual development. The common mycoparasitic fungus Trichoderma atroviride is one of the most widely studied agents of biological control of plant-pathogenic fungi that also serves as a model for the research on regulation of asexual sporulation (conidiation) by environmental stimuli such as light and/or mechanical injury. In order to learn the possible involvement of LAE1 in these two traits, we assessed the effect of deletion and overexpression of lae1 gene on conidiation and mycoparasitic interaction. In the presence of light, conidiation was 50% decreased in a Δ lae1 and 30-50% increased in lae1-overexpressing (OElae1) strains. In darkness, Δ lae1 strains did not sporulate, and the OElae1 strains produced as much spores as the parent strain. Loss-of-function of lae1 also abolished sporulation triggered by mechanical injury of the mycelia. Deletion of lae1 also increased the sensitivity of T. atroviride to oxidative stress, abolished its ability to defend against other fungi and led to a loss of mycoparasitic behaviour, whereas the OElae1 strains displayed enhanced mycoparasitic vigor. The loss of mycoparasitic activity in the Δ lae1 strain correlated with a significant underexpressionn of several genes normally upregulated during mycoparasitic interaction (proteases, GH16 ß-glucanases, polyketide synthases and small cystein-rich secreted proteins), which in turn was reflected in the partial reduction of formation of fungicidal water soluble metabolites and volatile compounds. Our study shows T. atroviride LAE1 is essential for asexual reproduction in the dark and for defense and parasitism on other fungi.

Figure 1. Asexual sporulation of T. atroviride.

(A) Quantitation of conidiation of the parent (P1), Δ lae1-1 and OElae1 strains on PDA in light (white bars) and in darkness (full bars). Values are means of at least three independent biological experiments. Similar investigations with strain Δ lae1-2 yielded values within ±8% of those of Δ lae1-1. All values are statistically different by the students t-test (p<0.05).

Figure 2. Effect of LAE1, and the blue light receptors BLR1 and BLR2 on each others expression: (A) Expression of blr1 (dark grey) and blr2 (light grey) in Δlae1-1 and OElae1 in light (L) and darkness (D); (B) expression of lae1 in Δblr1 and Δblr2 in light (L) and darkness (D).

Vertical bars indicate the standard deviation (N ≥3). Expression of lae1, blr1 and blr2 was normalized to the expression of tef1. Relative gene expression is calculated as the ratio of the normalized expression in the mutant in –fold of that of the parent strain P1. None of the difference was found to be statistically relevant by students t-test (p>0.15).

Figure 3. Effect of hydrogen peroxide on growth of T. atroviride parent strain and lae1 mutant strains.

Growth on PDA was monitored in intervals of 6–12 hrs for a period of up to 100 hrs. Growth rates were calculated from the phase where the increase in colony diameter vs time was linear and were calculated as mm/h. In the figure, the growth rate of the strain in the absence of hydrogen peroxide was set to 100%, and all other growth rates related to it. Data are means from at least 8 independent biological replicas.

Figure 4. Phenotype of confrontation of T. atroviride P1 and thelae1 mutants OElae1 and Δlae1 (all T) against B. cinerea (B), A. alternata (A) and R. solani (R) after termination of growth of the latter three fungi.

Left plates are photographed from the backside, right plates are photographed from top. (B) Test for production of WSC: T. atroviride parent strain, and the Δ lae1-1 and OElae1 mutants were grown on PDA agar covered by cellophane, and then removed and Alternaria alternata (Aa), Rhizoctonia solani (Rs) and Sclerotinia sclerotiorum (Ss) placed on these plates. The plates were photographed after 7 days.

Figure 5. Modulation of expression of genes putatively involved in mycoparasitism (A).

Ratios of expression between the parent strain and either the Δ lae1-1 (blue bars) or the OElae1 strain (red bars) are shown. The * and Δ symbols indicate p<0.05. Genes are given by their Triat2 number: 34350, GH16 ß-glycosidase; 144038, aspartyl protease; 160930, aspartyl protease; 128831, C-type lectin; 132795, C-type lectin; 134073, cyanovirin-N; 156014, GH16 ß-1,3/ß-1,4-glucanase; 85006, polyketide synthase (PKS); 53332, small cystein-rich secreted protein (SSCP); 131539, SSCP; 145909, subtilisin-like protease; 149951, subtilisin-like protease. Data are plotted relative to wild-type (P1) control. (B) Expression of the lipoxygenase gene in the Δ lae1 and OElae1 strains in light (L) and darkness (D). (C) Growth of A. alternata in the presence of VOC from the T. atroviride parent strain and the lae1 mutants. Only R. solani is shown but essentially similar results were also obtained with R. solani and B. cinerea. Single plates from several (N>4) experiments are shown.