Analysis of two in planta expressed LysM effector homologs from the fungus Mycosphaerella graminicola reveals novel functional properties and varying contributions to virulence on wheat

Abstract

Secreted effector proteins enable plant pathogenic fungi to manipulate host defenses for successful infection. Mycosphaerella graminicola causes Septoria tritici blotch disease of wheat (Triticum aestivum) leaves. Leaf infection involves a long (approximately 7 d) period of symptomless intercellular colonization prior to the appearance of necrotic disease lesions. Therefore, M. graminicola is considered as a hemibiotrophic (or necrotrophic) pathogen. Here, we describe the molecular and functional characterization of M. graminicola homologs of Ecp6 (for extracellular protein 6), the Lysin (LysM) domain-containing effector from the biotrophic tomato (Solanum lycopersicum) leaf mold fungus Cladosporium fulvum, which interferes with chitin-triggered immunity in plants. Three LysM effector homologs are present in the M. graminicola genome, referred to as Mg3LysM, Mg1LysM, and MgxLysM. Mg3LysM and Mg1LysM genes were strongly transcriptionally up-regulated specifically during symptomless leaf infection. Both proteins bind chitin; however, only Mg3LysM blocked the elicitation of chitin-induced plant defenses. In contrast to C. fulvum Ecp6, both Mg1LysM and Mg3LysM also protected fungal hyphae against plant-derived hydrolytic enzymes, and both genes show significantly more nucleotide polymorphism giving rise to nonsynonymous amino acid changes. While Mg1LysM deletion mutant strains of M. graminicola were fully pathogenic toward wheat leaves, Mg3LysM mutant strains were severely impaired in leaf colonization, did not trigger lesion formation, and were unable to undergo asexual sporulation. This virulence defect correlated with more rapid and pronounced expression of wheat defense genes during the symptomless phase of leaf colonization. These data highlight different functions for MgLysM effector homologs during plant infection, including novel activities that distinguish these proteins from C. fulvum Ecp6.

Figure 1.

Sequence alignments of the three putative LysM effector homologs from M. graminicola. A, Mg1LysM, MgxLysM, and Mg3LysM aligned against CfEcp6. Black boxes highlight conserved amino acids (identical and conservative changes); gray boxes highlight similar amino acids. B, Alignment of individual LysM domains from all four proteins. C, Phylogram with distance indicator showing the relatedness of the LysM domains from the four proteins.

Figure 2.

Gene expression analysis of the three MgLysM effector homologs during in vitro and in planta growth. A, Diagrammatic representation of the structures of the three MgLysM genes, highlighting the positions of PCR primers (horizontal arrows) subsequently used to determine whether the predicted genes were expressed. Black bars indicate the predicted exons present in the gene model. Note that the primers used for the analysis of MgxLysM structure and expression reside in the predicted 5′ upstream and 3′ downstream regions for this gene. The genomic sequence length in bp is given for each gene as an indication of scale. B, Testing of the various MgLysM PCR primers on various templates including fungal genomic DNA (lanes 1), cDNA isolated from fungus growing on PDB (lanes 2), and cDNA isolated from infected wheat leaves 8 d after fungal inoculation (lanes 3). End-point PCR was performed for 40 cycles for each primer pair, and the data presented do not report relative expression levels. C, Real-time RT-PCR gene expression analysis during growth in vitro (PDB and CDB) and a time course of plant infection. The various phases of leaf infection are indicated. Gene expression data are presented relative to the expression of the fungal β-tubulin gene. The figure highlights that Mg1LysM and Mg3LysM genes are both strongly expressed during symptomless plant infection, whereas MgxLysM is not; these findings are representative of duplicate experiments with similar results.

Figure 3.

Both the Mg1LysM and Mg3LysM proteins bind chitin but only Mg3LysM suppresses chitin-induced elicitation of tomato suspension-cultured cells. A, The purified MgLysM proteins were incubated in the presence of the insoluble carbohydrates chitin, crab shell chitin and chitosan, and the plant-derived carbohydrates xylan and cellulose. Following centrifugation, both the pellet (P) and the supernatant (S) were analyzed on protein gels. Both the Mg3LysM and Mg1LysM proteins were pelleted only in the presence of insoluble chitin. B, Tomato cell culture chitin-induced medium alkalinization assays in the presence and absence of Mg3LysM and Mg1LysM proteins. Only Mg3LysM is able to suppress elicitation of the cells by 10 nm chitin (Gn6). Graphs represent an average of two to four measurements in all cases.

Figure 4.

Mg1LysM and Mg3LysM protect fungal hyphae from hydrolysis by plant hydrolytic enzymes. Micrographs of T. viride taken 4 to 6 h after addition of water, crude extract of tomato leaves containing intracellular, hydrolytic enzymes including basic chitinases (ChiB), pretreatment with 30 μm CfAvr4 followed by addition of tomato extract (Avr4 + ChiB), pretreatment with 30 μm CfEcp6 followed by addition of tomato extract (Ecp6 + ChiB), pretreatment with 30 μm Mg3LysM followed by addition of tomato extract (Mg3LysM + ChiB), and pretreatment with 30 μm Mg1LysM followed by addition of tomato extract (Mg1LysM + ChiB). A representative figure from three independent experiments is shown. Bars = 10 μm.

Figure 5.

Allelic variation in the Mg1LysM and Mg3LysM genes. A and B, Mg1LysM (A) and Mg3LysM (B) genes were sequenced from genomic DNA of a nine-member differential isolate set varying in their interactions with wheat STB disease resistance genes. Sites with synonymous nucleotide substitutions are marked with T symbols. Sites with nonsynonymous substitutions contributing to amino acid changes are marked with flags. C and D, The posterior probability of a site in the Mg1LysM (C) and Mg3LysM (D) coding sequences evolving under positive selective pressure. Sites with high posterior probabilities are considered likely to evolve under strong positive selection. The gray horizontal bars indicate the positions of the encoded LysM domains. The figure highlights that, in contrast to CfEcp6, both MgLysM genes show more evidence of sequence evolution between fungal isolates.

Figure 6.

Gene disruption strains of Mg3LysM (ΔMg3LysM) are severely impaired in their ability to colonize and reproduce in wheat leaves. A, Wheat leaves inoculated with the wild-type strain of M. graminicola photographed 21 d after inoculation at three different fungal spore concentrations. B, Wheat leaves inoculated with a ΔMg3LysM mutant (ΔMg3LysM-2) strain photographed 21 d after inoculation at three different spore concentrations. C, Leaf infected with wild-type fungus at 30 d post inoculation highlighting the presence of abundant fungal sporulation structures (black foci). D, Leaf infected with ΔMg3LysM mutant fungus at 30 d post inoculation. E, Quantitative PCR on DNA isolated from fungus-infected leaves to determine the levels of fungal biomass during colonization by the wild-type (WT) and ΔMg3LysM mutant strains. Data are provided for two independent ΔMg3LysM mutants (ΔMg3LysM-2 and ΔMg3LysM-3). The data are shown relative to biomass detected at 6 d after inoculation for the wild-type fungus and are representative of three independent experiments performed on each strain.

Figure 7.

The ΔMg3LysM mutants trigger more rapid and strong expression of wheat defense-related genes during the early symptomless phase of the interaction. RNA was isolated from wheat leaves inoculated with either the wild type or ΔMg3LysM mutants across the symptomless period of infection. Real-time RT-PCR was performed on the wheat PR1 and Chitinase genes. Gene expression data are normalized to expression of the wheat β-tubulin gene and presented relative to that detected at day 0. The data are representative of duplicate experiments with similar results.