MAPK cascades mediating Trichoderma brevicrassum strain TC967 against phytopathogen Rhizoctonia solani

Trichoderma brevicrassum strain TC967 is a novel biological control agent (BCA) against the plant pathogen Rhizoctonia solani and promotes plant growth. MAPK (mitogen-activated protein kinase) cascades were involved in biocontrol by Trichoderma, but functions of each MAPK in regulating biocontrol have not been characterized in one Trichoderma. In this study, we assembled and annotated the genome of strain TC967, and identified its three MAPK gene sequences. Functions of Fus3-, Slt2- and Hog1-MAPK in strain TC967 were dissected. The three MAPKs were all involved in hyphal growth. The Hog1-MAPK was essential for conidiation and tolerance to hyperosmotic stress. The Fus3- and Slt2-MAPK both mediated cell-wall integrity (CWI) and activities of chitinase and protease. The Fus3- and Hog1-MAPK mediated response to oxidative stress. Our biocontrol assays demonstrated that the Fus3- and Slt2-MAPK mutants were considerably more effective in disease control than the wild-type strain. RNA-seq analysis revealed that MAPK collectively played a major role in regulating biocontrol-related gene expressions, including of the genes in charge of secondary metabolism, fungal cell wall-degrading enzymes (FCWDEs) and small secreted cysteine-rich proteins (SSCPs). Author summary Soil-born fungal pathogens pose an emerging threat to crop production. Trichoderma brevicrassum strain TC967 has the ability to control the notorious phytopathogen Rhizoctonia solani and promote plant growth. In this study, we explored the functions of three-types of MAPK in mediating biocontrol process, and uncovered that Fus3-, Slt2- and Hog1-MAPK are involved in hyphal growth, conidiation, tolerance to hyperosmotic stress, cell-wall integrity, activities of chitinase and protease, and response to oxidative stress. Biocontrol ability of strain TC967 was accelerated after deletion of the Fus3- and Slt2-MAPK genes. MAPK collectively played a major role in regulating biocontrol-related gene expressions as revealed by RNA-seq analysis. To our knowledge, this is the first report of the functions of MAPKs in regulating biocontrol in one Trichoderma. Our results provide a reference for improvement of biocontrol ability of Trichoderma strains from the view of MAPK cascades.

The mapk deletion mutants were characterized for their defects in developments.
150 All the mapk mutants showed significant slower growth rates compared with the wild 151 and complemented mutant strains. However, the growth rate of Fus3-MAPK gene 152 mutants (Δtbmk1-6 and Δtbmk1-8) were faster than that of Slt2-MAPK gene (Δtbmk2-153 4 and Δtbmk2-5) and Hog1-MAPK gene (Δtbmk3-2 and Δtbmk3-19) mutants ( Fig 3A   154 and B). The hyphal morphology has changed after deletion of mapk genes. The mutants 155 of Fus3-MAPK and Hog1-MAPK genes produced more branches at the hyphal tips 156 with a normal width. The vegetative hyphae of Slt2-MAPK gene deletion mutants had 157 thinner branches at the hyphal tips than that of the wild and complemented mutant 158 strains (Fig 3). The results indicate that Slt2-and Hog1-MAPK are more critical for 159 vegetative growth than Fus3-MAPK.

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To evaluate the involvement of mapk genes in conidiation, all the mutants and 161 wild strain were cultured on PDA medium supplied with cycle light for 7 days. Our 162 examinations showed that deletion of Hog1-MAPK gene led to seriously impaired 163 conidiation. The Hog1-MAPK gene mutants (Δtbmk3-2 and Δtbmk3-19) almost totally 164 lost the ability of producing conidia (Fig 4A and C). The Fus3-MAPK gene (Δtbmk1-6 165 and Δtbmk1-8) and the Slt2-MAPK gene (Δtbmk2-4 and Δtbmk2-5) mutants did not 166 significantly influence conidial production. Microscopic observations of the samples 167 revealed that the conidiophores of Hog1-MAPK gene mutants were degenerated, which 168 were much thinner and fewer, and conidia formed were rather sparse (Fig 4B). We also 9 169 found that the Fus3-and Slt2-MAPK gene mutants lost concentric rings in colony, but 170 the wild-type and complemented strains produced obvious concentric rings on PDA 171 medium supplied with cycle light. In order to find out whether the mapk mutants are 172 light dependence for conidiation, all strains were kept in a dark incubator. As the result, 173 conidiation of Slt2-MAPK gene mutants (Δtbmk2-4 and Δtbmk2-5) were sustained 174 under dark condition (Fig 4D). In addition, Slt2-MAPK gene mutants produced 175 massively conidia in submerged cultures (48 h) (Fig 4E), whereas no conidia were 176 detected in cultures of the other mutants and wild strain even after 7 days. These facts 177 suggest that Hog1-MAPK regulates conidiation, and Slt2-MAPK participates positive 178 regulation of photoreception in the process of conidiation.
179 Responses to abiotic stresses of mapk mutants

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The cell-wall integrity of the wild and mapk mutant strains were investigated by 181 testing the sensitivity to cell wall-damaging agent. The wild and mapk mutant strains 182 were incubated on PDA medium containing Congo red (CR). The Fus3-and Slt2-183 MAPK gene mutants showed increased sensitivity to CR (Fig 5 Table).
307 Analysis of the DEGs of all FCWDEs in TC967, 12 genes were significantly changed 308 in the three mapk mutants compared with wild TC967 (FDR < 0.05). Seven genes were 309 upregulated in the three mapk mutants, including of one gene in chitinase family 310 (GH18), two in β-1,3-glucanase family (GH55), two in α-mannosidase family (GH92), 311 and one in acetyl xylan esterase family (CE4). Meanwhile, four genes were 312 downregulated in all the mutants, and respectively distributed in cell wall glucanase 313 (GH16), GH18, and fructose-bisphosphate aldolase (GH76) (Fig 10B) 319 The amino acids with small size (less than 300) and more than 4% content of cysteine 320 residues were chosen for analysis. A total of 261 SSCPs were predicted in strain TC967,  Table).   . The secondary metabolites produced by the 434 wild type TC967 did not show obvious inhibition effects on R. solani (Fig 6 A and B).
435 However, inhibition ability was promoted by the deletion of Slt2-MAPK gene (Fig 6 A   436 and B). The possible reason is that it changes the content of secondary metabolites. It 437 was also proved that most of the core genes related to secondary metabolism were 438 upregulated in Slt2-MAPK gene mutants (Fig 10A).  (Fig 7). The result of RNA-seq indicated that expression of the chitinase gene 452 EVM0010536 (ech42-like) was obviously upregulated, and expression of GH16 family 453 gene was downregulated in each of the three MAPK gene mutants (Fig 10B)  510 The colonies of mapk complement mutants were screened on PDA plates supplemented 511 with 1200 µg/mL neomycin sulphate. The resulting mutants were confirmed by PCR 512 assays, primers were listed in S5 Table. 513  Table)