The Putative Protein Methyltransferase LAE1 of <i>Trichoderma atroviride</i> Is a Key Regulator of Asexual Development and Mycoparasitism

<div><p>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 <i>Trichoderma atroviride</i> 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 <i>lae1</i> gene on conidiation and mycoparasitic interaction. In the presence of light, conidiation was 50% decreased in a <b>Δ</b><i>lae1</i> and 30–50% increased in <i>lae1</i>-overexpressing (OE<i>lae1</i>) strains. In darkness, <b>Δ</b><i>lae1</i> strains did not sporulate, and the OE<i>lae1</i> strains produced as much spores as the parent strain. Loss-of-function of <i>lae1</i> also abolished sporulation triggered by mechanical injury of the mycelia. Deletion of <i>lae1</i> also increased the sensitivity of <i>T. atroviride</i> to oxidative stress, abolished its ability to defend against other fungi and led to a loss of mycoparasitic behaviour, whereas the <i>OElae1</i> strains displayed enhanced mycoparasitic vigor. The loss of mycoparasitic activity in the <b>Δ</b><i>lae1</i> 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 <i>T. atroviride</i> LAE1 is essential for asexual reproduction in the dark and for defense and parasitism on other fungi.</p></div

Effect of LAE1, and the blue light receptors BLR1 and BLR2 on each others expression: (A) Expression of <i>blr1</i> (dark grey) and <i>blr2</i> (light grey) in Δ<i>lae1-1</i> and <i>OElae1</i> in light (L) and darkness (D); (B) expression of <i>lae1</i> in Δ<i>blr1</i> and Δ<i>blr2</i> in light (L) and darkness (D).

<p>Vertical bars indicate the standard deviation (N ≥3). Expression of <i>lae1, blr1</i> and <i>blr2</i> was normalized to the expression of <i>tef1</i>. 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).</p

Asexual sporulation of <i>T. atroviride</i>.

<p>(A) Quantitation of conidiation of the parent (P1), <b>Δ</b><i>lae1-1</i> and <i>OElae1</i> 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 <b>Δ</b><i>lae1-2</i> yielded values within ±8% of those of <b>Δ</b><i>lae1-1.</i> All values are statistically different by the students t-test (p<0.05).</p

Phenotype of confrontation of <i>T. atroviride</i> P1 and the<i>lae1</i> mutants <i>OElae1</i> and Δ<i>lae1</i> (all T) against <i>B. cinerea</i> (B), <i>A. alternata</i> (A) and <i>R. solani</i> (R) after termination of growth of the latter three fungi.

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

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

<p>Ratios of expression between the parent strain and either the <b>Δ</b><i>lae1-1</i> (blue bars) or the <i>OElae1</i> strain (red bars) are shown. The * and <b>Δ</b> 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 <b>Δ</b><i>lae1</i> and <i>OElae1</i> strains in light (L) and darkness (D). (C) Growth of <i>A. alternata</i> in the presence of VOC from the <i>T. atroviride</i> parent strain and the <i>lae1</i> mutants. Only <i>R. solani</i> is shown but essentially similar results were also obtained with <i>R. solani</i> and <i>B. cinerea.</i> Single plates from several (N>4) experiments are shown.</p

Effect of hydrogen peroxide on growth of <i>T. atroviride</i> parent strain and <i>lae1</i> mutant strains.

<p>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.</p

Figure 2

<p>(a) Growth comparison of QM 9414 to the Δ3405 strain on minimal media supplemented with D-glucose, lactose and D-galactose. Pictures were taken on day 4. (b-g) Comparison of strains QM 9414 (grey), Δ3405 (white) and <i>ptef::3405</i> (black) pregrown in minimal medium supplemented with 1% glycerol and,after washing of the mycelium extensivly with sterile water, transferred to minimal medium containing 1% lactose. (b) Accumulation of <i>cel6a</i>, <i>cel7a</i> and <i>xyr1</i> transcripts measured by qPCR. Samples were taken 5, 8, 16 and 24 h after replacement to 1% lactose. The expression is given in relation to <i>tef1</i> gene expression, where the <i>tef1</i> expression value equals one. Mean values ± SD of three independent experiments are shown. (c) Lactose concentration in the medium during growth of QM 9414 (filled diamonds), Δ3405 (empty circles) and <i>ptef::3405</i> (empty squares) on 1% lactose. (d) Biomass formation of strains QM 9414 (filled diamonds), Δ3405 (empty circles) and <i>ptef::3405</i> (empty squares) on 1% lactose. Mean values ± SD of three independent experiments are given. (e) SDS-PAGE analysis of culture filtrates from QM 9414, Δ3405 and <i>ptef::3405.</i> Samples taken at 16 and 24 h after replacement to lactose. (f) ß-galactosidase activity determined with <i>o</i>-nitrophenyl-ß-D-galactopyranoside as the substrate. Error bars indicate the standard deviation of three independent experiments.</p

The lactose-induced CAZome of <i>T. reesei.</i>

<p>The lactose-induced CAZome of <i>T. reesei.</i></p

Balance of significantly^* regulated transcripts during growth of <i>T. reesei</i> on lactose.

*<p>either>or <2-fold vs. glucose and glycerol.</p>*<p>%-values refer to the total number of genes.</p>**<p>%-values refer to the category of genes (e.g. identified, unknown or unique, resp.).</p

Genes involved in sexual development that are upregulated in <i>T. reesei</i> during growth on lactose.

*<p>fold change.</p