Zhongqiang Qi

Eight RGS and RGS-like proteins orchestrate growth, differentiation, and pathogenicity of Magnaporthe oryzae.

A previous study identified MoRgs1 as an RGS protein that negative regulates G-protein signaling to control developmental processes such as conidiation and appressorium formation in Magnaporthe oryzae. Here, we characterized additional seven RGS and RGS-like proteins (MoRgs2 through MoRgs8). We found that MoRgs1 and MoRgs4 positively regulate surface hydrophobicity, conidiation, and mating. Indifference to MoRgs1, MoRgs4 has a role in regulating laccase and peroxidase activities. MoRgs1, MoRgs2, MoRgs3, MoRgs4, MoRgs6, and MoRgs7 are important for germ tube growth and appressorium formation. Interestingly, MoRgs7 and MoRgs8 exhibit a unique domain structure in which the RGS domain is linked to a seven-transmembrane motif, a hallmark of G-protein coupled receptors (GPCRs). We have also shown that MoRgs1 regulates mating through negative regulation of Gα MoMagB and is involved in the maintenance of cell wall integrity. While all proteins appear to be involved in the control of intracellular cAMP levels, only MoRgs1, MoRgs3, MoRgs4, and MoRgs7 are required for full virulence. Taking together, in addition to MoRgs1 functions as a prominent RGS protein in M. oryzae, MoRgs4 and other RGS and RGS-like proteins are also involved in a complex process governing asexual/sexual development, appressorium formation, and pathogenicity.

R-SNARE Homolog MoSec22 Is Required for Conidiogenesis, Cell Wall Integrity, and Pathogenesis of Magnaporthe oryzae

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular vesicle fusion, which is an essential cellular process of the eukaryotic cells. To investigate the role of SNARE proteins in the rice blast fungus Magnaporthe oryzae, MoSec22, an ortholog of Saccharomyces cerevisiae SNARE protein Sec22, was identified and the MoSEC22 gene disrupted. MoSec22 restored a S. cerevisiae sec22 mutant in resistance to cell wall perturbing agents, and the ΔMosec22 mutant also exhibited defects in mycelial growth, conidial production, and infection of the host plant. Treatment with oxidative stress inducers indicated a breach in cell wall integrity, and staining and quantification assays suggested abnormal chitin deposition on the lateral walls of hyphae of the ΔMosec22 mutant. Furthermore, hypersensitivity to the oxidative stress correlates with the reduced expression of the extracellular enzymes peroxidases and laccases. Our study thus provides new evidence on the conserved function of Sec22 among fungal organisms and indicates that MoSec22 has a role in maintaining cell wall integrity affecting the growth, morphogenesis, and virulence of M. oryzae.

MoVam7, a Conserved SNARE Involved in Vacuole Assembly, Is Required for Growth, Endocytosis, ROS Accumulation, and Pathogenesis of Magnaporthe oryzae

Soluble NSF attachment protein receptor (SNARE) proteins play a central role in membrane fusion and vesicle transport of eukaryotic organisms including fungi. We previously identified MoSce22 as a homolog of Saccharomyces cerevisiae SNARE protein Sec22 to be involved in growth, stress resistance, and pathogenicity of Magnaporthe oryzae. Here, we provide evidences that MoVam7, an ortholog of S. cerevisiae SNARE protein Vam7, exerts conserved functions in vacuolar morphogenesis and functions in pathogenicity of M. oryzae. Staining with neutral red and FM4-64 revealed the presence of abnormal fragmented vacuoles and an absence of the Spitzenkörper body in the ΔMovam7 mutant. The ΔMovam7 mutant also exhibited reduced vegetative growth, poor conidiation, and failure to produce the infection structure appressorium. Additionally, treatments with cell wall perturbing agents indicated weakened cell walls and altered distributions of the cell wall component chitin. Furthermore, the ΔMovam7 mutant showed a reduced accumulation of reactive oxygen species (ROS) in the hyphal apex and failed to cause diseases on the rice plant. In summary, our studies indicate that MoVam7, like MoSec22, is a component of the SNARE complex whose functions in vacuole assembly also underlies the growth, conidiation, appressorium formation, and pathogenicity of M. oryzae. Further studies of MoVam7, MoSec22, and additional members of the SNARE complex are likely to reveal critical mechanisms in vacuole formation and membrane trafficking that is linked to fungal pathogenicity.

MoSwi6, an APSES family transcription factor, interacts with MoMps1 and is required for hyphal and conidial morphogenesis, appressorial function and pathogenicity of Magnaporthe oryzae.

The Magnaporthe oryzae mitogen-activated protein kinase (MAPK) MoMps1 plays a critical role in the regulation of various developmental processes, including cell wall integrity, stress responses and pathogenicity. To identify potential effectors of MoMps1, we characterized the function of MoSwi6, a homologue of Saccharomyces cerevisiae Swi6 downstream of MAPK Slt2 signalling. MoSwi6 interacted with MoMps1 both in vivo and in vitro, suggesting a possible functional link analogous to Swi6-Slt2 in S. cerevisiae. Targeted gene disruption of MoSWI6 resulted in multiple developmental defects, including reduced hyphal growth, abnormal formation of conidia and appressoria, and impaired appressorium function. The reduction in appressorial turgor pressure also contributed to an attenuation of pathogenicity. The ΔMoswi6 mutant also displayed a defect in cell wall integrity, was hypersensitive to oxidative stress, and showed a significant reduction in transcription and activity of extracellular enzymes, including peroxidases and laccases. Collectively, these roles are similar to those of MoMps1, confirming that MoSwi6 functions in the MoMps1 pathway to govern growth, development and full pathogenicity.

The actin-regulating kinase homologue MoArk1 plays a pleiotropic function in Magnaporthe oryzae

Summary Endocytosis is an essential cellular process in eukaryotic cells that involves concordant functions of clathrin and adaptor proteins, various protein and lipid kinases, phosphatases and the actin cytoskeleton. In Saccharomyces cerevisiae, Ark1p is a member of the serine/threonine protein kinase (SPK) family that affects profoundly the organization of the cortical actin cytoskeleton. To study the function of MoArk1, an Ark1p homologue identified in Magnaporthe oryzae, we disrupted the MoARK1 gene and characterized the ΔMoark1 mutant strain. The ΔMoark1 mutant exhibited various defects ranging from mycelial growth and conidial formation to appressorium‐mediated host infection. The ΔMoark1 mutant also exhibited decreased appressorium turgor pressure and attenuated virulence on rice and barley. In addition, the ΔMoark1 mutant displayed defects in endocytosis and formation of the Spitzenkörper, and was hyposensitive to exogenous oxidative stress. Moreover, a MoArk1‐green fluorescent protein (MoArk1‐GFP) fusion protein showed an actin‐like localization pattern by localizing to the apical regions of hyphae. This pattern of localization appeared to be regulated by the N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins MoSec22 and MoVam7. Finally, detailed analysis revealed that the proline‐rich region within the MoArk1 serine/threonine kinase (S_TKc) domain was critical for endocytosis, subcellular localization and pathogenicity. These results collectively suggest that MoArk1 exhibits conserved functions in endocytosis and actin cytoskeleton organization, which may underlie growth, cell wall integrity and virulence of the fungus.

Pleiotropic Function of the Putative Zinc-Finger Protein MoMsn2 in Magnaporthe oryzae

The mitogen-activated protein kinase MoOsm1-mediated osmoregulation pathway plays crucial roles in stress responses, asexual and sexual development, and pathogenicity in Magnaporthe oryzae. Utilizing an affinity purification approach, we identified the putative transcriptional activator MoMsn2 as a protein that interacts with MoOsm1 in vivo. Disruption of the MoMSN2 gene resulted in defects in aerial hyphal growth, conidial production, and infection of host plants. Quantitative reverse transcription-polymerase chain reaction analysis showed that the expression of several genes involved in conidiophore formation was reduced in ΔMomsn2, suggesting that MoMsn2 might function as a transcriptional regulator of these genes. Subsequently, MoCos1 was identified as one of the MoMsn2 targets through yeast one-hybrid analysis in which MoMsn2 binds to the AGGGG and CCCCT motif of the MoCOS1 promoter region. Phenotypic characterization showed that MoMsn2 was required for appressorium formation and penetration and pathogenicity. Although the ΔMomsn2 mutant was tolerant to the cell-wall stressor Calcofluor white, it was sensitive to common osmotic stressors. Further analysis suggests that MoMsn2 is involved in the regulation of the cell-wall biosynthesis pathway. Finally, transcriptome data revealed that MoMsn2 modulates numerous genes participating in conidiation, infection, cell-wall integrity, and stress response. Collectively, our results led to a model in which MoMsn2 mediates a series of downstream genes that control aerial hyphal growth, conidiogenesis, appressorium formation, cell-wall biosynthesis, and infection and that also offer potential targets for the development of new disease management strategies.

The Function of MoGlk1 in Integration of Glucose and Ammonium Utilization in Magnaporthe oryzae

Hexokinases are conserved proteins functioning in glucose sensing and signaling. The rice blast fungus Magnaporthe oryzae contains several hexokinases, including MoHxk1 (hexokinase) and MoGlk1 (glucokinase) encoded respectively by MoHXK1 and MoGLK1 genes. The heterologous expression of MoGlk1 and MoHxk1 in Saccharomyces cerevisiae confirmed their conserved functions. Disruption of MoHXK1 resulted in growth reduction in medium containing fructose as the sole carbon source, whereas disruption of MoGLK1 did not cause the similar defect. However, the ΔMoglk1 mutant displayed decreased proton extrusion and a lower biomass in the presence of ammonium, suggesting a decline in the utilization of ammonium. Additionally, the MoGLK1 allele lacking catalytic activity restored growth to the ΔMoglk1 mutant. Moreover, the expression of MoPMA1 encoding a plasma membrane H(+)-ATPase decreased in the ΔMoglk1 mutant that can be suppressed by glucose and G-6-P. Thus, MoGlk1, but not MoHxk1, regulates ammonium utilization through a mechanism that is independent from its catalytic activity.

Genome plasticity in filamentous plant pathogens contributes to the emergence of novel effectors and their cellular processes in the host

Plant diseases cause extensive yield loss of crops worldwide, and secretory ‘warfare’ occurs between plants and pathogenic organisms all the time. Filamentous plant pathogens have evolved the ability to manipulate host processes and facilitate colonization through secreting effectors inside plant cells. The stresses from hosts and environment can drive the genome dynamics of plant pathogens. Remarkable advances in plant pathology have been made owing to these adaptable genome regions of several lineages of filamentous phytopathogens. Characterization new effectors and interaction analyses between pathogens and plants have provided molecular insights into the plant pathways perturbed during the infection process. In this mini-review, we highlight promising approaches of identifying novel effectors based on the genome plasticity. We also discuss the interaction mechanisms between plants and their filamentous pathogens and outline the possibilities of effector gene expression under epigenetic control that will be future directions for research.

Orotate phosphoribosyl transferase MoPyr5 is involved in uridine 5′-phosphate synthesis and pathogenesis of Magnaporthe oryzae

Orotate phosphoribosyl transferase (OPRTase) plays an important role in de novo and salvage pathways of nucleotide synthesis and is widely used as a screening marker in genetic transformation. However, the function of OPRTase in plant pathogens remains unclear. In this study, we characterized an ortholog of Saccharomyces cerevisiae Ura5, the OPRTase MoPyr5, from the rice blast fungus Magnaporthe oryzae. Targeted gene disruption revealed that MoPyr5 is required for mycelial growth, appressorial turgor pressure and penetration into plant tissues, invasive hyphal growth, and pathogenicity. Interestingly, the ∆Mopyr5 mutant is also involved in mycelial surface hydrophobicity. Exogenous uridine 5′-phosphate (UMP) restored vegetative growth and rescued the defect in pathogenicity on detached barley and rice leaf sheath. Collectively, our results show that MoPyr5 is an OPRTase for UMP biosynthesis in M. oryzae and indicate that UTP biosynthesis is closely linked with vegetative growth, cell wall integrity, and pathogenicity of fungus. Our results also suggest that UMP biosynthesis would be a good target for the development of novel fungicides against M. oryzae.

MoMyb1 is required for asexual development and tissue-specific infection in the rice blast fungus Magnaporthe oryzae.

BackgroundThe Myb super-family of proteins contain a group of functionally diverse transcriptional activators found in plant, animal and fungus. Myb proteins are involved in cell proliferation, differentiation and apoptosis, and have crucial roles in telomeres. The purpose of this study was to characterize the biological function of Myb1 protein in the rice blast fungus Magnaporthe oryzae.ResultsWe identified the Saccharomyces cerevisiae BAS1 homolog MYB1 in M. oryzae, named MoMyb1. MoMyb1 encodes a protein of 322 amino acids and has two SANT domains and is well conserved in various organisms. Targeted gene deletion of MoMYB1 resulted in a significant reduction in vegetative growth and showed defects in conidiation and conidiophore development. Quantitative RT-PCR analysis revealed that the transcription levels of several conidiophore-related genes were apparently decreased in the ΔMomyb1 mutant. Inoculation with mycelia mats displayed that the virulence of the ΔMomyb1 mutant was not changed on rice leaves but was non-pathogenic on rice roots in comparison to the wild type Guy11. In addition, ∆Momyb1 mutants showed increased resistance to osmotic stresses but more sensitive to cell wall stressor calcofluor white (CFW). Further analysis revealed that MoMyb1 has an important role in the cell wall biosynthesis pathway.ConclusionThis study provides the evidence that MoMyb1 is a key regulator involved in conidiogenesis, stress response, cell wall integrity and pathogenesis on rice roots in the filamentous phytopathogen M. oryzae.