LysM Proteins Regulate Fungal Development and Contribute to Hyphal Protection and Biocontrol Traits in Clonostachys rosea

Abstract

Lysin motif (LysM) modules are approximately 50 amino acids long and bind to peptidoglycan, chitin and its derivatives. Certain LysM proteins in plant pathogenic and entomopathogenic fungi are shown to scavenge chitin oligosaccharides and thereby dampen host defense reactions. Other LysM proteins can protect the fungal cell wall against hydrolytic enzymes. In this study, we investigated the biological function of LysM proteins in the mycoparasitic fungus Clonostachys rosea. The C. rosea genome contained three genes coding for LysM-containing proteins and gene expression analysis revealed that lysm1 and lysm2 were induced during mycoparasitic interaction with Fusarium graminearum and during colonization of wheat roots. Lysm1 was suppressed in germinating conidia, while lysm2 was induced during growth in chitin or peptidoglycan-containing medium. Deletion of lysm1 and lysm2 resulted in mutants with increased levels of conidiation and conidial germination, but reduced ability to control plant diseases caused by F. graminearum and Botrytis cinerea. The Δlysm2 strain showed a distinct, accelerated mycelial disintegration phenotype accompanied by reduced biomass production and hyphal protection against hydrolytic enzymes including chitinases, suggesting a role of LYSM2 in hyphal protection against chitinases. The Δlysm2 and Δlysm1Δlysm2 strains displayed reduced ability to colonize wheat roots, while only Δlysm1Δlysm2 failed to suppress expression of the wheat defense response genes PR1 and PR4. Based on our data, we propose a role of LYSM1 as a regulator of fungal development and of LYSM2 in cell wall protection against endogenous hydrolytic enzymes, while both are required to suppress plant defense responses. Our findings expand the understanding of the role of LysM proteins in fungal-fungal interactions and biocontrol.

Keywords: Clonostachys rosea; LysM effector; LysM protein; antagonism; biocontrol; biological control; fungal–fungal interactions; mycoparasitism.

FIGURE 1

Non-synonymous nucleotide substitution in the lysm1, lysm2, and chiC2 genes in C. rosea strains. LYSM1 has one LysM module between amino acid positions 127–171; LYSM2 and CHIC2 contain two LysM modules between amino acid positions 235–280, 289–339, and 309–354, 372–419, respectively. The single nucleotide polymorphism (SNPs) were compared among worldwide collection of 53 C. rosea strains. The number in parenthesis indicates the number of strains share the same nucleotide polymorphism. Detailed information of all SNPs reported in lysm1, lysm2, and chiC2 can be found in Supplementary Table S2.

FIGURE 2

Expression analysis of lysm1 and lysm2 in C. rosea. (A) During in vitro interaction with B. cinerea (Cr-Bc) or F. graminearum (Cr-Fg) at two time points: at contact (C) and at 24 h after contact (AC) stage, and wheat root (Cr-Wr) at 48 and 96 hpi. C. rosea confronted with itself (Cr-Cr) was used as control. (B) Gene expression in self (Cr Culfil) or F. graminearum (Fg Culfil) culture filtrates, mycelia grown in liquid Czapek-dox (CZ) was used as control. (C) Gene expression analysis in C. rosea mycelia grown in liquid SMS medium amended with 1% colloidal chitin or 0.1% peptidoglycan (Pep) as a sole carbon source. SMS medium amended with 1% glucose was used as control. (D) Gene expression analysis during conidial germination. Liquid SMS with 1% glucose was used for the conidial germination. C. rosea mycelia grown in SMS with 1% glucose was used as control. Relative expression level based on RT-qPCR was calculated as the ratio between the target lysm gene and β-tubulin gene using 2^–ΔΔCt method (Livak and Schmittgen, 2001), and compared with the respective control. Error bar represent standard deviation based on three or five biological replicates. Statistically significant differences (P ≤ 0.05) in gene expression between treatments were determined using Fisher’s exact test or T-test and are indicated by different letters.

FIGURE 3

Analysis of conidial germination and germ tube length 8 h post inoculation on half-strength PDA medium. (A,C) Frequency of germinating conidia was determined by counting the number of germinating and non-germinating conidia. (B,C) Germ tube length was measured with the ImageJ image analysis software (Schneider et al., 2012). Scale bar: 10 μm. Error bars represent standard deviation based on four biological replicates. Different letters indicate statistically significant differences (P ≤ 0.05) within the experiments based on Fisher’s exact test.

FIGURE 4

Phenotypic characterizations of C. rosea WT, deletion and complementation strains. (A,B) Deletion of lysm2 affects hyphal protection. After 2 weeks of inoculation no visible difference in mycelial disintegration was found between the WT and the deletion strains. However, after 12 weeks of inoculation, mycelial disintegration was distinct in the Δlysm2 and Δlysm1Δlysm2 deletion strains. (C) Conidiation of WT, lysm deletion and complementation strains on PDA medium after 2 and 12 weeks of inoculation. The number of conidia from each treatment was normalized to their respective mycelial biomass. (D–F) Deletion of lysm1 or lysm2 affects the mycelial pellet formation in liquid media. (G) Biomass production in C. rosea WT and deletion strains 12 weeks post inoculation. Biomass production was determined measuring mycelial dry weight. (H) Chitinase activity analysis in culture filtrates from C. rosea WT and the deletion strains 2 weeks, 7 weeks and 12 wpi in liquid SMS medium with 1% glucose. Error bars represent standard deviation based on five biological replicates. Different letters indicate statistically significant differences based on Tukey HSD method at the 95% significance level.

FIGURE 5

Cell wall protection assay of C. rosea strains against snailase enzyme mix. Protoplast release from C. rosea and deletion strains was counted after 60 min of incubation. The experiment was performed in five replicates. Different letters indicate statistically significant differences based on Fisher’s exact test at the 95% significance level.

FIGURE 6

Mycoparasitism analyses of C. rosea (right side in the plate) strains against B. cinerea (left side in the plate). (A) The Δlysm2 and Δlysm1Δlysm2 strains showed reduced ability to grow on B. cinerea. (B) Growth rate (overgrow) of C. rosea WT and deletion strains on B. cinerea. The growth of C. rosea on B. cinerea was measured from the point of mycelial contact. (C) Mycelial biomass of C. rosea WT and deletion strains 3 weeks post inoculation. Mycelial biomass was quantified by measuring the pixel intensity of the photographs using ImageJ image analysis software (Schneider et al., 2012). The experiment was performed in four biological replicates and photographs of representative plates were taken 3 weeks post inoculation.

FIGURE 7

In vivo bioassay to test the biocontrol ability of C. rosea strains against B. cinerea and F. graminearum. (A) Measurement of B. cinerea necrotic lesions on detached leaves of A. thaliana plants. The leaves were inoculated with C. rosea strains 60 min before application of B. cinerea and allowed to interact for 60 h. Only pathogen inoculated leaves were used as control. Necrotic lesion area was measured under the microscope using DeltaPix camera and software. The exprement was performed in five biological replicates with six leaves in each replicate in each treatment. (B) Foot rot disease on wheat using sand seedling test. Seedlings were harvested 3 weeks post inoculation and disease symptoms were scored on 0–4 scale. The experiment was performed in five biological replicates with 15 plants in each replicate. Different letters indicate statistically significant differences (P ≤ 0.05) within experiments based on the Fisher’s exact test.

FIGURE 8

Expression analysis of defense related genes and root colonization assay in wheat. (A) Gene expression analysis of defense response related genes in wheat roots colonized by C. rosea strains. Total RNA isolated from wheat roots 5 days post inoculation of WT and Δlysm strains and was used for RT-qPCR. Expression level of PR1 and PR4 genes was normalized to expression of the wheat β-tubulin (Marshall et al., 2011). Relative expression level based on RT-qPCR was calculated using 2^–ΔΔCt method (Livak and Schmittgen, 2001), and compared with the water inoculated control. (B) Determination of C. rosea root colonization in wheat roots, 5 days post inoculation, by quantifying DNA level using RT-qPCR. C. rosea colonization is expressed as the ratio between C. rosea DNA and wheat DNA. For DNA quantification actin and Hor1 were used as target gene for C. rosea and wheat, respectively. (C) Internal root colonization by C. rosea strains. Surface sterilized roots were homogenized in phosphate buffer and serial dilutions were plated on Rose Bengal selection plates under sterile condition at 25°C. Colony forming units (cfus) were counted 3 days post plating. Statistically significant differences (P ≤ 0.05) in gene expression between treatments were determined using Fisher’s exact test and are indicated by different letters. Error bars represent standard deviation based on five biological replicates.