MoWhi2 Mediates Mitophagy to Regulate Conidiation and Pathogenesis in Magnaporthe oryzae

Mitophagy refers to the specific process of degrading mitochondria, which is an important physiological process to maintain the balance of mitochondrial quantity and quality in cells. At present, the mechanisms of mitophagy in pathogenic fungi remain unclear. Magnaporthe oryzae (Syn. Pyricularia oryzae), the causal agent of rice blast disease, is responsible for the most serious disease of rice. In M. oryzae, mitophagy occurs in the foot cells and invasive hyphae to promote conidiation and infection. In this study, fluorescent observations and immunoblot analyses showed that general stress response protein MoWhi2 is required for mitophagy in M. oryzae. In addition, the activation of the autophagy, pexophagy and cytoplasm-to-vacuole targeting (CVT) pathway upon nitrogen starvation was determined using the GFP-MoATG8, GFP-SRL and MoAPE1-GFP strains and the ΔMowhi2 mutant in these backgrounds. The results indicated that MoWhi2 is specifically required for mitophagy in M. oryzae. Further studies showed that mitophagy in the foot cells and invasive hyphae of the ΔMowhi2 was interrupted, leading to reduced conidiation and virulence in the ΔMowhi2 mutant. Taken together, we found that MoWhi2 contributes to conidiation and invasive growth by regulating mitophagy in M. oryzae.     

A Putative Effector CcSp84 of Cytospora chrysosperma Localizes to the Plant Nucleus to Trigger Plant Immunity

Cytospora chrysosperma is the main causal agent of poplar canker disease in China, especially in some areas with poor site conditions. Pathogens secrete a large number of effectors to interfere the plant immunity and promote their infection and colonization. Nevertheless, the roles of effectors in C. chrysosperma remain poorly understood. In this study, we identified and functionally characterized a candidate effector CcSp84 from C. chrysosperma, which contained a nuclear localization signal motif at the C-terminal and was highly induced during infection stages. Transient expression of CcSp84 in Nicotiana benthamiana leaves could trigger cell death. Additionally, deletion of CcSp84 significantly reduced fungal virulence to the polar twigs, while no obvious defects were observed in fungal growth and sensitivity to H₂O₂. Confocal microscopy revealed that CcSp84 labeled with a green fluorescent protein (GFP) was mainly accumulated in the plant nucleus. Further analysis revealed that the plant nucleus localization of CcSp84 was necessary to trigger plant immune responses, including ROS accumulation, callose deposition, and induced expression of jasmonic acid and ethylene defense-related genes. Collectively, our results suggest that CcSp84 is a virulence-related effector, and plant nucleus localization is required for its functions.     

Chloroplast-Expressed MSI-99 in Tobacco Improves Disease Resistance and Displays Inhibitory Effect against Rice Blast Fungus

Rice blast is a major destructive fungal disease that poses a serious threat to rice production and the improvement of blast resistance is critical to rice breeding. The antimicrobial peptide MSI-99 has been suggested as an antimicrobial peptide conferring resistance to bacterial and fungal diseases. Here, a vector harboring the MSI-99 gene was constructed and introduced into the tobacco chloroplast genome via particle bombardment. Transformed plants were obtained and verified to be homoplastomic by PCR and Southern hybridization. In planta assays demonstrated that the transgenic tobacco plants displayed an enhanced resistance to the fungal disease. The evaluation of the antimicrobial activity revealed that the crude protein extracts from the transgenic plants manifested an antimicrobial activity against E. coli, even after incubation at 120 °C for 20 min, indicating significant heat stability of MSI-99. More importantly, the MSI-99-containing protein extracts were firstly proved in vitro and in vivo to display significant suppressive effects on two rice blast isolates. These findings provide a strong basis for the development of new biopesticides to combat rice blast.     

Stress-Responsive Expression,Subcellular Localization and Protein–Protein Interactions of the Rice Metacaspase Family

Metacaspases, a class of cysteine-dependent proteases like caspases in animals, are important regulators of programmed cell death (PCD) during development and stress responses in plants. The present study was focused on comprehensive analyses of expression patterns of the rice metacaspase (OsMC) genes in response to abiotic and biotic stresses and stress-related hormones. Results indicate that members of the OsMC family displayed differential expression patterns in response to abiotic (e.g., drought, salt, cold, and heat) and biotic (e.g., infection by Magnaporthe oryzae, Xanthomonas oryzae pv. oryzae and Rhizoctonia solani) stresses and stress-related hormones such as abscisic acid, salicylic acid, jasmonic acid, and 1-amino cyclopropane-1-carboxylic acid (a precursor of ethylene), although the responsiveness to these stresses or hormones varies to some extent. Subcellular localization analyses revealed that OsMC1 was solely localized and OsMC2 was mainly localized in the nucleus. Whereas OsMC3, OsMC4, and OsMC7 were evenly distributed in the cells, OsMC5, OsMC6, and OsMC8 were localized in cytoplasm. OsMC1 interacted with OsLSD1 and OsLSD3 while OsMC3 only interacted with OsLSD1 and that the zinc finger domain in OsMC1 is responsible for the interaction activity. The systematic expression and biochemical analyses of the OsMC family provide valuable information for further functional studies on the biological roles of OsMCs in PCD that is related to abiotic and biotic stress responses.     

Secondary Metabolites of the Rice Blast Fungus Pyricularia oryzae: Biosynthesis and Biological Function

Plant pathogenic fungi produce a wide variety of secondary metabolites with unique and complex structures. However, most fungal secondary metabolism genes are poorly expressed under laboratory conditions. Moreover, the relationship between pathogenicity and secondary metabolites remains unclear. To activate silent gene clusters in fungi, successful approaches such as epigenetic control, promoter exchange, and heterologous expression have been reported. Pyricularia oryzae, a well-characterized plant pathogenic fungus, is the causal pathogen of rice blast disease. P. oryzae is also rich in secondary metabolism genes. However, biosynthetic genes for only four groups of secondary metabolites have been well characterized in this fungus. Biosynthetic genes for two of the four groups of secondary metabolites have been identified by activating secondary metabolism. This review focuses on the biosynthesis and roles of the four groups of secondary metabolites produced by P. oryzae. These secondary metabolites include melanin, a polyketide compound required for rice infection; pyriculols, phytotoxic polyketide compounds; nectriapyrones, antibacterial polyketide compounds produced mainly by symbiotic fungi including endophytes and plant pathogens; and tenuazonic acid, a well-known mycotoxin produced by various plant pathogenic fungi and biosynthesized by a unique NRPS-PKS enzyme.     

Genome-Wide Association Study Identifies a Rice Panicle Blast Resistance Gene,Pb2, Encoding NLR Protein

Rice blast is one of the main diseases in rice and can occur in different rice growth stages. Due to the complicated procedure of panicle blast identification and instability of panicle blast infection influenced by the environment, most cloned rice resistance genes are associated with leaf blast. In this study, a rice panicle blast resistance gene, Pb2, was identified by genome-wide association mapping based on the panicle blast resistance phenotypes of 230 Rice Diversity Panel 1 (RDP1) accessions with 700,000 single-nucleotide polymorphism (SNP) markers. A genome-wide association study identified 18 panicle blast resistance loci (PBRL) within two years, including 9 reported loci and 2 repeated loci (PBRL2 and PBRL13, PBRL10 and PBRL18). Among them, the repeated locus (PBRL10 and PBRL18) was located in chromosome 11. By haplotype and expression analysis, one of the Nucleotide-binding domain and Leucine-rich Repeat (NLR) Pb2 genes was highly conserved in multiple resistant rice cultivars, and its expression was significantly upregulated after rice blast infection. Pb2 encodes a typical NBS-LRR protein with NB-ARC domain and LRR domain. Compared with wild type plants, the transgenic rice of Pb2 showed enhanced resistance to panicle and leaf blast with reduced lesion number. Subcellular localization of Pb2 showed that it is located on plasma membrane, and GUS tissue-staining observation found that Pb2 is highly expressed in grains, leaf tips and stem nodes. The Pb2 transgenic plants showed no difference in agronomic traits with wild type plants. It indicated that Pb2 could be useful for breeding of rice blast resistance.     

Overexpression of Peroxisome-Localized GmABCA7 Promotes Seed Germination in Arabidopsis thaliana

Peroxisome is one of the important organelles for intracellular lipid metabolism in plant cells and β-oxidation of fatty acids in peroxisomes provides the energy for oil-containing seed germination. In this study, we identified an ATP-binding cassette (ABC) transporter gene, GmABCA7 from soybean, which is highly expressed in the different developmental stages of seeds. Transient expression of GmABCA7 in tobacco epidermal cells showed that GmABCA7 was specifically localized at the peroxisomes. Overexpression of GmABCA7 in Arabidopsis does not change seed phenotypes, or the overall levels of lipid, protein and sugar stored in the seeds; however, the transgenic seeds produced more gluconeogenic pathway precursors such as succinate and malate and germinated earlier compared to the wild type seeds. These results suggest that GmABCA7 may affect the β-oxidation of fatty acids and play an important role in seed germination.     

Fusarium graminearum Infection Strategy in Wheat Involves a Highly Conserved Genetic Program That Controls the Expression of a Core Effectome

Fusarium graminearum, the main causal agent of Fusarium Head Blight (FHB), is one of the most damaging pathogens in wheat. Because of the complex organization of wheat resistance to FHB, this pathosystem represents a relevant model to elucidate the molecular mechanisms underlying plant susceptibility and to identify their main drivers, the pathogen’s effectors. Although the F. graminearum catalog of effectors has been well characterized at the genome scale, in planta studies are needed to confirm their effective accumulation in host tissues and to identify their role during the infection process. Taking advantage of the genetic variability from both species, a RNAseq-based profiling of gene expression was performed during an infection time course using an aggressive F. graminearum strain facing five wheat cultivars of contrasting susceptibility as well as using three strains of contrasting aggressiveness infecting a single susceptible host. Genes coding for secreted proteins and exhibiting significant expression changes along infection progress were selected to identify the effector gene candidates. During its interaction with the five wheat cultivars, 476 effector genes were expressed by the aggressive strain, among which 91% were found in all the infected hosts. Considering three different strains infecting a single susceptible host, 761 effector genes were identified, among which 90% were systematically expressed in the three strains. We revealed a robust F. graminearum core effectome of 357 genes expressed in all the hosts and by all the strains that exhibited conserved expression patterns over time. Several wheat compartments were predicted to be targeted by these putative effectors including apoplast, nucleus, chloroplast and mitochondria. Taken together, our results shed light on a highly conserved parasite strategy. They led to the identification of reliable key fungal genes putatively involved in wheat susceptibility to F. graminearum, and provided valuable information about their putative targets.