Serine protease CrKP43 interacts with MAPK and regulates fungal development and mycoparasitism in Clonostachys chloroleuca.

IMPORTANCE: Mycoparasites play important roles in the biocontrol of plant fungal diseases, during which they secret multiple hydrolases such as serine proteases to degrade their fungal hosts. In this study, we demonstrated that the serine protease CrKP43 was involved in C. chloroleuca development and mycoparasitism with the regulation of Crmapk. To the best of our knowledge, it is the first report on the functions and regulatory mechanisms of serine proteases in C. chloroleuca. Our findings will provide new insight into the regulatory mechanisms of serine proteases in mycoparasites and contribute to clarifying the mechanisms underlying mycoparasitism of C. chloroleuca, which will facilitate the development of highly efficient fungal biocontrol agents as well.

Pre-treatment with Dazomet enhances the biocontrol efficacy of purpureocillium lilacinum to Meloidogyne incognita.

BACKGROUND: Meloidogyne incognita greatly restricts the production of protected vegetables in China. Application of biocontrol agent Purpureocillium lilacinum is an important practice to control the nematode; however, instability usually occurs especially in heavily infested field. This study aimed to illustrate the high efficiency of P. lilacinum agent with fumigant Dazomet in vitro. RESULTS: P. lilacinum YES-2-14 showed strong parasitic and nematicidal activities to M. incognita. Pre-treatment with Dazomet significantly enhanced the biocontrol effects of the fungus. After fumigation with Dazomet at a dosage of 7.5 mg kg- 1 soil, parasitism of YES-2-14 on M. incognita eggs increased by more than 50%. Meanwhile, when P. lilacinum fermentation filtrate treated following Dazomet fumigation at 10 and 20 mg kg- 1 soil, the mortalities of second-stage juveniles (J2s) increased by 110.2% and 72.7%, respectively. Both Dazomet and P. lilacinum significantly reduced the penetration ability of J2s to tomato roots. When P. lilacinum filtrate used alone, the J2s penetrating into the young roots decreased by 48.8% at 4 dpi; while in the combined treatment, almost no J2 was detected within the roots at 4 dpi and the number of knots reduced by more than 99% at 45 dpi, indicating a synergistic effect of the biocontrol fungus and fumigant. CONCLUSIONS: Pre-treatment with Dazomet greatly increased the biocontrol efficacy of P. lilacinum to M. incognita. This research provides insight into the efficient management of plant parasitic nematodes and effective use of biocontrol agents.

Glucose-6-phosphate 1-Epimerase CrGlu6 Contributes to Development and Biocontrol Efficiency in Clonostachys chloroleuca.

Clonostachys chloroleuca (formerly classified as C. rosea) is an important mycoparasite active against various plant fungal pathogens. Mitogen-activated protein kinase (MAPK) signaling pathways are vital in mycoparasitic interactions; they participate in responses to diverse stresses and mediate fungal development. In previous studies, the MAPK-encoding gene Crmapk has been proven to be involved in mycoparasitism and the biocontrol processes of C. chloroleuca, but its regulatory mechanisms remain unclear. Aldose 1-epimerases are key enzymes in filamentous fungi that generate energy for fungal growth and development. By protein-protein interaction assays, the glucose-6-phosphate 1-epimerase CrGlu6 was found to interact with Crmapk, and expression of the CrGlu6 gene was significantly upregulated when C. chloroleuca colonized Sclerotinia sclerotiorum sclerotia. Gene deletion and complementation analyses showed that CrGlu6 deficiency caused abnormal morphology of hyphae and cells, and greatly reduced conidiation. Moreover, deletion mutants presented much lower antifungal activities and mycoparasitic ability, and control efficiency against sclerotinia stem rot was markedly decreased. When the CrGlu6 gene was reinserted, all biological characteristics and biocontrol activities were recovered. These findings provide new insight into the mechanisms of glucose-6-phosphate 1-epimerase in mycoparasitism and help to further reveal the regulation of MAPK and its interacting proteins in the biocontrol of C. chloroleuca.

Monitoring Mycoparasitism of Clonostachys rosea against Botrytis cinerea Using GFP.

Clonostachys rosea is an important mycoparasite, with great potential for controlling numerous plant fungal diseases. Understanding the mechanisms and modes of action will assist the development and application of this biocontrol fungus. In this study, the highly efficient C. rosea 67-1 strain was marked with the green fluorescent protein (GFP), and the transformant possessed the same biological characteristics as the wild-type strain. Fungal interactions with Botrytis cinerea during co-culture and encounter on tomato leaves were assessed by fluorescence confocal and electron microscopy. The results indicated that once the two fungi met, the hyphae of C. rosea grew alongside those of B. cinerea, then attached tightly to the host and developed special structures, via which the biocontrol fungus penetrated the host and absorbed nutrients, eventually disintegrating the cells of the pathogen. Mycoparasitism to B. cinerea was also observed on tomato leaves, suggesting that C. rosea can colonize on plants and act following the invasion of the pathogenic fungus.

Screening and characterisation of proteins interacting with the mitogen-activated protein kinase Crmapk in the fungus Clonostachys chloroleuca.

Clonostachys chloroleuca 67-1 (formerly C. rosea 67-1) is a promising mycoparasite with great potential for controlling various plant fungal diseases. The mitogen-activated protein kinase (MAPK)-encoding gene Crmapk is of great importance to the mycoparasitism and biocontrol activities of C. chloroleuca. To investigate the molecular mechanisms underlying the role of Crmapk in mycoparasitism, a high-quality yeast two hybrid (Y2H) library of C. chloroleuca 67-1 was constructed, and proteins interacting with Crmapk were characterised. The library contained 1.6 × 107 independent clones with a recombination rate of 96%, and most inserted fragments were > 1 kb. The pGBKT7-Crmapk bait vector with no self-activation or toxicity to yeast cells was used to screen interacting proteins from the Y2H library, resulting in 60 candidates, many linked to metabolism, cellular processes and signal transduction. Combined bioinformatics and transcriptome analyses of C. chloroleuca 67-1 and ΔCrmapk mutant mycoparasitising Sclerotinia sclerotiorum sclerotia, 41 differentially expressed genes were identified, which might be the targets of the Fus3/Kss1-MAPK pathway. The results provide a profile of potential protein interactions associated with MAPK enzymes in mycoparasites, and are of great significance for understanding the mechanisms of Crmapk regulating C. chloroleuca mycoparasitism.

Tandem Mass Tag labelling quantitative acetylome analysis of differentially modified proteins during mycoparasitism of Clonostachys chloroleuca 67-1.

Lysine acetylation (Kac) is an important post-translational modification (PTM) of proteins in all organisms, but its functions have not been extensively explored in filamentous fungi. In this study, a Tandem Mass Tag (TMT) labelling lysine acetylome was constructed, and differentially modified Kac proteins were quantified during mycoparasitism and vegetative growth in the biocontrol fungus Clonostachys chloroleuca 67-1, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1448 Kac sites were detected on 740 Kac proteins, among which 126 sites on 103 proteins were differentially regulated. Systematic bioinformatics analyses indicate that the modified Kac proteins were from multiple subcellular localizations and involved in diverse functions including chromatin assembly, glycometabolism and redox activities. All Kac sites were characterized by 10 motifs, including the novel CxxKac motif. The results suggest that Kac proteins may have effects of broadly regulating protein interaction networks during C. chloroleuca parasitism to Sclerotinia sclerotiorum sclerotia. This is the first report of a correlation between Kac events and the biocontrol activity of C. chloroleuca. Our findings provide insight into the molecular mechanisms underlying C. chloroleuca control of plant fungal pathogens regulated by Kac proteins.

Cell Wall Biogenesis Protein Phosphatase CrSsd1 Is Required for Conidiation, Cell Wall Integrity, and Mycoparasitism in Clonostachys rosea.

Cell wall biogenesis protein phosphatases play important roles in various cellular processes in fungi. However, their functions in the widely distributed mycoparasitic fungus Clonostachys rosea remain unclear, as do their potential for controlling plant fungal diseases. Herein, the function of cell wall biogenesis protein phosphatase CrSsd1 in C. rosea 67-1 was investigated using gene disruption and complementation approaches. The gene-deficient mutant ΔCrSsd1 exhibited much lower conidiation, hyphal growth, mycoparasitic ability, and biocontrol efficacy than the wild-type (WT) strain, and it was more sensitive to sorbitol and Congo red. The results indicate that CrSsd1 is involved in fungal conidiation, osmotic stress adaptation, cell wall integrity, and mycoparasitism in C. rosea.

Involvement of BcYak1 in the Regulation of Vegetative Differentiation and Adaptation to Oxidative Stress of Botrytis cinerea.

Yak1, a member of the dual-specificity tyrosine phosphorylation-regulated protein kinases, plays an important role in diverse cellular processes in fungi. However, to date, the role of BcYak1 in Botrytis cinerea, the causal agent of gray mold diseases in various plant species, remains uncharacterized. Our previous study identified one lysine acetylation site (Lys252) in BcYak1, which is the first report of such a site in Yak1. In this study, the function of BcYak1 and its lysine acetylation site were investigated using gene disruption and site-directed mutagenesis. The gene deletion mutant ΔBcYak1 not only exhibits much lower pathogenicity, conidiation and sclerotium formation, but was also much more sensitive to H2O2 and the ergosterol biosynthesis inhibitor (EBI) triadimefon. The Lys252 site-directed mutagenesis mutant strain ΔBcYak1-K252Q (mimicking the acetylation of the site), however, only showed lower sclerotium formation and higher sensitivity to H2O2. These results indicate that BcYAK1 is involved in the vegetative differentiation, adaptation to oxidative stress and triadimefon, and virulence of B. cinerea.

Acetylome analysis reveals the involvement of lysine acetylation in diverse biological processes in Phytophthora sojae.

Lysine acetylation is a dynamic and highly conserved post-translational modification that plays an important regulatory role in almost every aspects of cell metabolism in both eukaryotes and prokaryotes. Phytophthora sojae is one of the most important plant pathogens due to its huge economic impact. However, to date, little is known about the functions of lysine acetylation in this Phytopthora. Here, we conducted a lysine acetylome in P. sojae. Overall, 2197 lysine acetylation sites in 1150 proteins were identified. The modified proteins are involved in diverse biological processes and are localized to multiple cellular compartments. Importantly, 7 proteins involved in the pathogenicity or the secretion pathway of P. sojae were found to be acetylated. These data provide the first comprehensive view of the acetylome of P. sojae and serve as an important resource for functional analysis of lysine acetylation in plant pathogens.

Proteome-wide analysis of lysine acetylation in the plant pathogen Botrytis cinerea.

Lysine acetylation is a dynamic and reversible post-translational modification that plays an important role in diverse cellular processes. Botrytis cinerea is the most thoroughly studied necrotrophic species due to its broad host range and huge economic impact. However, to date, little is known about the functions of lysine acetylation in this plant pathogen. In this study, we determined the lysine acetylome of B. cinerea through the combination of affinity enrichment and high-resolution LC-MS/MS analysis. Overall, 1582 lysine acetylation sites in 954 proteins were identified. Bioinformatics analysis shows that the acetylated proteins are involved in diverse biological functions and show multiple cellular localizations. Several particular amino acids preferred near acetylation sites, including K(ac)Y, K(ac)H, K(ac)***R, K(ac)F, FK(ac) and K(ac)***K, were identified in this organism. Protein interaction network analysis demonstrates that a variety of interactions are modulated by protein acetylation. Interestingly, 6 proteins involved in virulence of B. cinerea, including 3 key components of the high-osmolarity glycerol pathway, were found to be acetylated, suggesting that lysine acetylation plays regulatory roles in pathogenesis. These data provides the first comprehensive view of the acetylome of B. cinerea and serves as a rich resource for functional analysis of lysine acetylation in this plant pathogen.

Acetylome analysis reveals the involvement of lysine acetylation in biosynthesis of antibiotics in Bacillus amyloliquefaciens.

Lysine acetylation is a major post-translational modification that plays an important regulatory role in almost every aspects in both eukaryotes and prokaryotes. Bacillus amyloliquefaciens, a Gram-positive bacterium, is very effective for the control of plant pathogens. However, very little is known about the function of lysine acetylation in this organism. Here, we conducted the first lysine acetylome in B. amyloliquefaciens through a combination of highly sensitive immune-affinity purification and high-resolution LC-MS/MS. Overall, we identified 3268 lysine acetylation sites in 1254 proteins, which account for 32.9% of the total proteins in this bacterium. Till date, this is the highest ratio of acetylated proteins that have been identified in bacteria. Acetylated proteins are associated with a variety of biological processes and a large fraction of these proteins are involved in metabolism. Interestingly, for the first time, we found that about 71.1% (27/38) and 78.6% (22/28) of all the proteins tightly related to the synthesis of three types of pepketides and five families of lipopeptides were acetylated, respectively. These findings suggest that lysine acetylation plays a critical role in the regulation of antibiotics biosynthesis. These data serves as an important resource for further elucidation of the physiological role of lysine acetylation in B. amyloliquefaciens.