The CfMK1 Gene Regulates Reproduction, Appressorium Formation, and Pathogenesis in a Pear Anthracnose-Causing Fungus

Colletotrichum fructicola, the causal agent of pear anthracnose, causes significant annual economic losses. Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways that play a crucial role in mediating cellular responses to environmental and host signals in plant pathogenic fungi. In this study, we identified an ortholog of the FUS3/KSS1-related MAPK gene, CfMK1, and characterized its function in C. fructicola. The Cfmk1 deletion mutants exhibited poorly developed aerial hyphae, autolysis, no conidial mass or perithecia on solid plates. However, the conidiation of the Cfmk1 mutant in PDB liquid medium was normal compared with that of the wild type (WT). Conidia of the Cfmk1 mutant exhibited a reduced germination rate on glass slides or plant surfaces. The Cfmk1 deletion mutants were unable to form appressoria and lost the capacity to penetrate plant epidermal cells. The ability of the Cfmk1 mutants to infect pear leaves and fruit was severely reduced. Moreover, RNA sequencing (RNA-seq) analysis of the WT and Cfmk1 mutant was performed, and the results revealed 1886 upregulated and 1554 downregulated differentially expressed genes (DEGs) in the mutant. The DEGs were significantly enriched in cell wall and pathogenesis terms, which was consistent with the defects of the Cfmk1 mutant in cell wall integrity and plant infection. Overall, our data demonstrate that CfMK1 plays critical roles in the regulation of aerial hyphal growth, asexual and sexual reproduction, autolysis, appressorium formation, and pathogenicity.

Host-Induced Gene Silencing of a Multifunction Gene Sscnd1 Enhances Plant Resistance Against Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a devastating necrotrophic fungal pathogen and has a substantial economic impact on crop production worldwide. Magnaporthe appressoria-specific (MAS) proteins have been suggested to be involved in the appressorium formation in Magnaporthe oryzae. Sscnd1, an MAS homolog gene, is highly induced at the early infection stage of S. sclerotiorum. Knock-down the expression of Sscnd1 gene severely reduced the virulence of S. sclerotiorum on intact rapeseed leaves, and their virulence was partially restored on wounded leaves. The Sscnd1 gene-silenced strains exhibited a defect in compound appressorium formation and cell integrity. The instantaneous silencing of Sscnd1 by tobacco rattle virus (TRV)-mediated host-induced gene silencing (HIGS) resulted in a significant reduction in disease development in tobacco. Three transgenic HIGS Arabidopsis lines displayed high levels of resistance to S. sclerotiorum and decreased Sscnd1 expression. Production of specific Sscnd1 siRNA in transgenic HIGS Arabidopsis lines was confirmed by stem-loop qRT-PCR. This study revealed that the compound appressorium-related gene Sscnd1 is required for cell integrity and full virulence in S. sclerotiorum and that Sclerotinia stem rot can be controlled by expressing the silencing constructs of Sscnd1 in host plants.

PoRal2 Is Involved in Appressorium Formation and Virulence via Pmk1 MAPK Pathways in the Rice Blast Fungus Pyricularia oryzae

Pyricularia oryzae is an important plant pathogenic fungus that can severely damage rice and wheat crops, leading to significant reductions in crop productivity. To penetrate into and invade tissues of its plant host, this fungus relies on an invasive structure known as an appressorium. Appressorium formation is rigorously regulated by the cAMP-PKA and Pmk1 MAPK pathways. Here, we identified PoRal2, a homologous protein of Schizosaccharomyces pombe Ral2, and characterized its roles in fungal development and virulence in P. oryzae. PoRal2 contains N-terminal kelch repeats and C-terminal BTB domains. PoRal2 is involved in sporulation, aerial hypha and conidiophore differentiation, appressorium formation, plant penetration, and virulence. During appressorium formation, ∆Poral2 mutants generate appressoria with long germ tubes on hydrophobic surfaces. ∆Poral2 mutants exhibited a defective response to exogenous cAMP and the activated RAS2G18V on a hydrophilic surface, indicating impairment in the cAMP-PKA or Pmk1 MAPK signaling pathways. Deletion of PoRAL2 leads to lowered Pmk1 phosphorylation level in the mutant. Moreover, PoRal2 is found to interact with Scd1, Smo1, and Mst50, which are involved in activation of Pmk1. In addition, the expression levels of MPG1, WISH, and PDEH in the cAMP-PKA pathway, RAS2 in both the cAMP-PKA and Pmk1 MAPK pathways, and melanin biosynthesis genes (ALB1, BUF1, and RSY1) were significantly down-regulated in the ∆Poral2. Therefore, PoRal2 is involved in fungal development and virulence by its crosstalk in the cAMP-PKA and Pmk1 MAPK signaling pathways.

Alternative Splicing of MoPTEN Is Important for Growth and Pathogenesis in Magnaporthe oryzae

Human PTEN, a dual-phosphatase tumor suppressor, is frequently dysregulated by alternative splicing. Fungi harbor PTEN homologs, but alternative splicing of fungal PTENs has not been reported as far as we know. Here, we described an alternative splicing case in the PTEN homolog of Magnaporthe oryzae (MoPTEN). Two splice variants of MoPTEN were detected and identified, which are resulted from an intron retention and exclusion (MoPTEN-1/2). Both proteins were different in lipid and protein phosphatase activity and in expression patterns. The MoPTEN deletion mutant (ΔMoPTEN) showed the defects in conidiation, appressorium formation, and pathogenesis. ΔMoPTEN could be completely restored by MoPTEN, but rescued partially by MoPTEN-1 in the defect of conidium and appressorium formation, and by MoPTEN-2 in the defect of invasive development. Assays to assess sensitivity to oxidative stress reveal the involvement of MoPTEN-2 in scavenging exogenous and host-derived H2O2. Taken together, MoPTEN undergoes alternative splicing, and both variants cooperatively contribute to conidium and appressorium development, and invasive hyphae growth in plant cells, revealing a novel disease development pathway in M. oryzae.

AaPKAc Regulates Differentiation of Infection Structures Induced by Physicochemical Signals From Pear Fruit Cuticular Wax, Secondary Metabolism, and Pathogenicity of Alternaria alternata

Alternaria alternata, the casual agent of black rot of pear fruit, can sense and respond to the physicochemical cues from the host surface and form infection structures during infection. To evaluate the role of cyclic AMP-dependent protein kinase (cAMP-PKA) signaling in surface sensing of A. alternata, we isolated and functionally characterized the cyclic adenosine monophosphate-dependent protein kinase A catalytic subunit gene (AaPKAc). Gene expression results showed that AaPKAc was strongly expressed during the early stages of appressorium formation on hydrophobic surfaces. Knockout mutants ΔAaPKAc were generated by replacing the target genes via homologous recombination events. We found that intracellular cAMP content increased but PKA content decreased in ΔAaPKAc mutant strain. Appressorium formation and infection hyphae were reduced in the ΔAaPKAc mutant strain, and the ability of the ΔAaPKAc mutant strain to recognize and respond to high hydrophobicity surfaces and different surface waxes was lower than in the wild type (WT) strain. In comparison with the WT strain, the appressorium formation rate of the ΔAaPKAc mutant strain on high hydrophobicity and fruit wax extract surface was reduced by 31.6 and 49.3% 4 h after incubation, respectively. In addition, AaPKAc is required for the hypha growth, biomass, pathogenicity, and toxin production of A. alternata. However, AaPKAc negatively regulated conidia formation, melanin production, and osmotic stress resistance. Collectively, AaPKAc is required for pre-penetration, developmental, physiological, and pathological processes in A. alternata.

CgEnd3 Regulates Endocytosis, Appressorium Formation, and Virulence in the Poplar Anthracnose Fungus Colletotrichum gloeosporioides

The hemibiotrophic ascomycete fungus Colletotrichum gloeosporioides is the causal agent of anthracnose on numerous plants, and it causes considerable economic losses worldwide. Endocytosis is an essential cellular process in eukaryotic cells, but its roles in C. gloeosporioides remain unknown. In our study, we identified an endocytosis-related protein, CgEnd3, and knocked it out via polyethylene glycol (PEG)-mediated protoplast transformation. The lack of CgEnd3 resulted in severe defects in endocytosis. C. gloeosporioides infects its host through a specialized structure called appressorium, and ΔCgEnd3 showed deficient appressorium formation, melanization, turgor pressure accumulation, penetration ability of appressorium, cellophane membrane penetration, and pathogenicity. CgEnd3 also affected oxidant adaptation and the expression of core effectors during the early stage of infection. CgEnd3 contains one EF hand domain and four calcium ion-binding sites, and it is involved in calcium signaling. A lack of CgEnd3 changed the responses to cell-wall integrity agents and fungicide fludioxonil. However, CgEnd3 regulated appressorium formation and endocytosis in a calcium signaling-independent manner. Taken together, these results demonstrate that CgEnd3 plays pleiotropic roles in endocytosis, calcium signaling, cell-wall integrity, appressorium formation, penetration, and pathogenicity in C. gloeosporioides, and it suggests that CgEnd3 or endocytosis-related genes function as promising antifungal targets.

Fungal jasmonate as a novel morphogenetic signal for pathogenesis

A key question that has remained unanswered is how pathogenic fungi switch from vegetative growth to infection-related morphogenesis during a disease cycle. Here, we identify a fungal oxylipin analogous to the well-known phytohormone jasmonic acid, as the principal morphogenesis signal responsible for such a developmental switch to pathogenicity in the rice-blast fungus Magnaporthe oryzae. We explored the molecular function(s) of such intrinsic jasmonic acid during pathogenic differentiation in M. oryzae via OPR1, which encodes a 12-Oxo-phytodienoic Acid Reductase essential for its biosynthesis. Loss of OPR1 led to prolonged vegetative growth, and a delayed initiation and improper development of infection structures in M. oryzae, reminiscent of phenotypes observed in mutants (e.g. pth11Δ and cpkAΔ) that are compromised for cyclic AMP signaling. Genetic- or chemical-complementation completely restored proper germ tube growth and appressorium formation in opr1Δ. Liquid chromatography mass spectrometry-based quantification revealed increased OPDA accumulation and a significant decrease in JA levels in the opr1Δ. Most interestingly, exogenous jasmonic acid also restored appressorium formation in the pth11Δ mutant that lacks G protein/cyclic AMP signaling. Epistasis analysis placed fungal jasmonate upstream of the cyclic AMP signaling in rice blast. Lastly, we show that intrinsic jasmonate orchestrates the cessation of vegetative phase and initiates pathogenic development via a regulatory interaction with the cyclic AMP cascade and redox signaling in rice blast.