MoRgs3 functions in intracellular reactive oxygen species perception-integrated cAMP signaling to promote appressorium formation in Magnaporthe oryzae.

Regulator of G-protein signaling (RGS) proteins exhibit GTPase-accelerating protein activities to govern G-protein function. In the rice blast fungus Magnaporthe oryzae, there is a family of at least eight RGS and RGS-like proteins (MoRgs1 to MoRgs8), each exhibiting distinct or shared functions in the growth, appressorium formation, and pathogenicity. MoRgs3 recently emerged as one of the crucial regulators that senses intracellular oxidation during appressorium formation. To explore this unique regulatory mechanism of MoRgs3, we identified the nucleoside diphosphate kinase MoNdk1 that interacts with MoRgs3. MoNdk1 phosphorylates MoRgs3 under induced intracellular reactive oxygen species levels, and MoRgs3 phosphorylation is required for appressorium formation and pathogenicity. In addition, we showed that MoRgs3 phosphorylation determines its interaction with MoCrn1, a coronin-like actin-binding protein homolog, which regulates MoRgs3 internalization. Finally, we provided evidence demonstrating that MoRgs3 functions in MoMagA-mediated cAMP signaling to regulate normal appressorium induction. By revealing a novel signal perception mechanism, our studies highlighted the complexity of regulation during the appressorium function and pathogenicity of the blast fungus. IMPORTANCE: We report that MoRgs3 becomes phosphorylated in an oxidative intracellular environment during the appressorium formation stage. We found that this phosphorylation is carried out by MoNdk1, a nucleoside diphosphate kinase. In addition, this phosphorylation leads to a higher binding affinity between MoRgs3 and MoCrn1, a coronin-like actin-binding protein that was implicated in the endocytic transport of several other RGS proteins of Magnaporthe oryzae. We further found that the internalization of MoRgs3 is indispensable for its GTPase-activating protein function toward the Gα subunit MoMagA. Importantly, we characterized how such cellular regulatory events coincide with cAMP signaling-regulated appressorium formation and pathogenicity in the blast fungus. Our studies uncovered a novel intracellular reactive oxygen species signal-transducing mechanism in a model pathogenic fungus with important basic and applied implications.

Targeting Magnaporthe oryzae effector MoErs1 and host papain-like protease OsRD21 interaction to combat rice blast.

Effector proteins secreted by plant pathogenic fungi are important artilleries against host immunity, but there is no precedent of such effectors being explored as antifungal targets. Here we demonstrate that MoErs1, a species-specific effector protein secreted by the rice blast fungus Magnaporthe oryzae, inhibits the function of rice papain-like cysteine protease OsRD21 involved in rice immunity. Disrupting MoErs1-OsRD21 interaction effectively controls rice blast. In addition, we show that FY21001, a structure-function-based designer compound, specifically binds to and inhibits MoErs1 function. FY21001 significantly and effectively controls rice blast in field tests. Our study revealed a novel concept of targeting pathogen-specific effector proteins to prevent and manage crop diseases.

Design and Synthesis of Novel Diphenyl Ether Carboxamide Derivatives To Control the Phytopathogenic Fungus Sclerotinia sclerotiorum.

Sclerotinia stem rot (SSR) caused by the phytopathogenic fungus Sclerotinia sclerotiorum has led to serious losses in the yields of oilseed rape and other crops every year. Here, we designed and synthesized a series of carboxamide derivatives containing a diphenyl ether skeleton by adopting the scaffold splicing strategy. From the results of the mycelium growth inhibition experiment, inhibition rates of compounds 4j and 4i showed more than 80% to control S. sclerotiorum at a dose of 50 µg/mL, which is close to that of the positive control (flubeneteram, 95%). Then, the results of a structure-activity relationship study showed that the benzyl scaffold was very important for antifungal activity and that introducing a halogen atom on the benzyl ring would improve antifungal activity. Furthermore, the results of an in vitro activity test suggested that these novel compounds can inhibit the activity of succinate dehydrogenase (SDH), and the binding mode of 4j with SDH was basically similar to that of the flutolanil derivative. Morphological observation of mycelium revealed that compound 4j could cause a damage on the mycelial morphology and cell structure of S. sclerotiorum, resulting in inhibition of the growth of mycelia. Furthermore, in vivo antifungal activity assessment of 4j displayed a good control of S. sclerotiorum (>97%) with a result similar to that of the positive control at a concentration of 200 mg/L. Thus, the diphenyl ether carboxamide skeleton is a new starting point for the discovery of new SDH inhibitors and is worthy of further development.

Predation of oomycetes by myxobacteria via a specialized CAZyme system arising from adaptive evolution.

As social micropredators, myxobacteria are studied for their abilities to prey on bacteria and fungi. However, their predation of oomycetes has received little attention. Here, we show that Archangium sp. AC19 secretes a carbohydrate-active enzyme (CAZyme) cocktail during predation on oomycetes Phytophthora. These enzymes include three specialized ß-1,3-glucanases (AcGlu13.1, -13.2 and -13.3) that act as a cooperative consortium to target ß-1,3-glucans of Phytophthora. However, the CAZymes showed no hydrolytic effects on fungal cells, even though fungi contain ß-1,3-glucans. Heterologous expression of AcGlu13.1, -13.2 or -13.3 enzymes in Myxococcus xanthus DK1622, a model myxobacterium that antagonizes but does not predate on P. sojae, conferred a cooperative and mycophagous ability that stably maintains myxobacteria populations as a mixture of engineered strains. Comparative genomic analyses suggest that these CAZymes arose from adaptive evolution among Cystobacteriaceae myxobacteria for a specific prey killing behavior, whereby the presence of Phytophthora promotes growth of myxobacterial taxa by nutrient release and consumption. Our findings demonstrate that this lethal combination of CAZymes transforms a non-predatory myxobacterium into a predator with the ability to feed on Phytophthora, and provides new insights for understanding predator-prey interactions. In summary, our work extends the repertoire of myxobacteria predatory strategies and their evolution, and suggests that these CAZymes can be engineered as a functional consortium into strains for biocontrol of Phytophothora diseases and hence crop protection.

MoIug4 is a novel secreted effector promoting rice blast by counteracting host OsAHL1-regulated ethylene gene transcription.

Magnaporthe oryzae secretes several effectors that modulate and hijack rice processes to colonize host cells, but the underlying mechanisms remain unclear. We report on a novel cytoplasmic effector MoIug4 that targets the rice ethylene pathway as a transcription repressor to subvert host immunity. We found that MoIug4 binds to the promoter of the host OsEIN2 gene that encodes a central signal transducer in the ethylene-signaling pathway. We also identified a MoIug4 interacting protein, OsAHL1, which acts as an AT-hook motif-containing protein binding to the A/T-rich promoter regions. Our knockout and overexpression studies showed that OsAHL1 positively regulates plant immunity in response to M. oryzae infection. OsAHL1 exhibits transcriptional regulatory activities by binding the OsEIN2 promoter region, similar to MoIug4. Intriguingly, we found that MoIug4 exhibits a higher binding affinity than OsAHL1 to the OsEIN2 promoter, suggesting differential regulatory specificities. These results revealed a counter-defense strategy by which the pathogen effector suppresses the activation of host defense genes by interfering with host transcription activator functions.

Transcriptomic Analysis of Extracellular RNA Governed by the Endocytic Adaptor Protein Cin1 of Cryptococcus deneoformans.

Membrane vesicles are considered virulence cargoes as they carry capsular and melanin components whose secretory transport is critical for the virulence of the human fungal pathogen Cryptococcus species. However, other components of the vesicles and their function in the growth and virulence of the fungus remain unclear. We have previously found that the cryptococcal intersectin protein Cin1 governs a unique Cin1-Wsp1-Cdc42 endocytic pathway required for intracellular transport and virulence. Using RNA sequencing, we compared the profiles of extracellular RNA (exRNA), including microRNA (miRNA), small interference RNA (siRNA), long noncoding RNA (lncRNA), and messenger RNA (mRNA) between the wild-type (WT), and derived Δcin1 mutant strains of Cryptococcus deneoformans. Seven hundred twelve miRNAs and 88 siRNAs were identified from WT, whereas 799 miRNAs and 66 siRNAs were found in Δcin1. Also, 572 lncRNAs and 7,721 mRNAs were identified from WT and 584 lncRNAs and 7,703 mRNAs from Δcin1. Differential expression analysis revealed that the disruption of CIN1 results in many important cellular changes, including those in exRNA expression, transport, and function. First, for miRNA target genes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that cellular processes, components, and macromolecular functions are the most affected pathways. A higher number of genes were involved in the intracellular transport of endocytosis. Second, the results of GO term and KEGG analysis of differentially expressed lncRNA target genes and mRNA genes were consistent with those of miRNA targets. In particular, protein export is the topmost affected pathway among lncRNA target genes and one of the affected pathways among mRNA genes. The result of quantitative real-time reverse transcription PCR (qRT-PCR) from 12 mRNAs tested is largely agreeable with that of RNA-Seq. Taken together, our studies provide a comprehensive reference that Cryptococcus secretes abundant RNAs and that Cin1 plays a critical role in regulating their secretion. Given the growing clinical importance of exRNAs, our studies illuminate the significance of exploring this cutting-edge technology in studies of cryptococcal pathogenesis for the discovery of novel therapeutic strategies.

Heat-Shock Proteins MoSsb1, MoSsz1, and MoZuo1 Attenuate MoMkk1-Mediated Cell-Wall Integrity Signaling and Are Important for Growth and Pathogenicity of Magnaporthe oryzae.

The mitogen-activated protein kinase (MAPK) MoMkk1 governs the cell-wall integrity (CWI) pathway in rice blast fungus Magnaporthe oryzae. To understand the underlying mechanism, we have identified MoSsb1 as one of the MoMkk1-interacting proteins. MoSsb1 is a stress-seventy subfamily B (Ssb) protein homolog, sharing high amino acid sequence homology with the 70-kDa heat shock proteins (Hsp70s). Hsp70 are a family of conserved and ubiquitously expressed chaperones that regulate protein biogenesis by promoting protein folding, preventing protein aggregation, and controlling protein degradation. We found that MoSsb1 regulates the synthesis of nascent polypeptide chains and this regulation is achieved by being in complex with other members of Hsp70s MoSsz1 and 40-kDa Hsp40 MoZuo1. MoSsb1 is important for the growth, conidiation, and full virulence of the blast fungus and this role is also shared by MoSsz1 and MoZuo1. Importantly, MoSsb1, MoSsz1, and MoZuo1 are all involved in the regulation of the CWI MAPK pathway by modulating MoMkk1 biosynthesis. Our studies reveal novel insights into how MoSsb1, MoSsz1, and MoZuo1 affect CWI signaling that is involved in regulating growth, differentiation, and virulence of M. oryzae and highlight the conserved functional mechanisms of heat-shock proteins in pathogenic fungi.

Activation of ethylene signaling pathways enhances disease resistance by regulating ROS and phytoalexin production in rice.

Ethylene plays diverse roles in plant growth, development and stress responses. However, the roles of ethylene signaling in immune responses remain largely unknown. In this study, we showed that the blast fungus Magnaporthe oryzae infection activated ethylene biosynthesis in rice. Resistant rice cultivars accumulated higher levels of ethylene than susceptible ones. Ethylene signaling components OsEIN2 and the downstream transcription factor OsEIL1 positively regulated disease resistance. Mutation of OsEIN2 led to enhanced disease susceptibility. Whole-genome transcription analysis revealed that responsive genes of ethylene, jasmonates (JAs) and reactive oxygen species (ROS) signaling as well as phytoalexin biosynthesis genes were remarkably induced. Transcription of OsrbohA/B, which encode NADPH oxidases, and OsOPRs, the JA biosynthesis genes, were induced by M. oryzae infection. Furthermore, we demonstrated that OsEIL1 binds to the promoters of OsrbohA/OsrbohB and OsOPR4 to activate their expression. These data suggest that OsEIN2-mediated OsrbohA/OsrbohB and OsOPR transcription may play essential roles in ROS generation, JA biosynthesis and the subsequent phytoalexin accumulation. Therefore, the involvement of ethylene signaling in disease resistance is probably by activation of ROS and phytoalexin production in rice during M. oryzae infection.

Global genome and transcriptome analyses of Magnaporthe oryzae epidemic isolate 98-06 uncover novel effectors and pathogenicity-related genes, revealing gene gain and lose dynamics in genome evolution.

Genome dynamics of pathogenic organisms are driven by pathogen and host co-evolution, in which pathogen genomes are shaped to overcome stresses imposed by hosts with various genetic backgrounds through generation of a variety of isolates. This same principle applies to the rice blast pathogen Magnaporthe oryzae and the rice host; however, genetic variations among different isolates of M. oryzae remain largely unknown, particularly at genome and transcriptome levels. Here, we applied genomic and transcriptomic analytical tools to investigate M. oryzae isolate 98-06 that is the most aggressive in infection of susceptible rice cultivars. A unique 1.4 Mb of genomic sequences was found in isolate 98-06 in comparison to reference strain 70-15. Genome-wide expression profiling revealed the presence of two critical expression patterns of M. oryzae based on 64 known pathogenicity-related (PaR) genes. In addition, 134 candidate effectors with various segregation patterns were identified. Five tested proteins could suppress BAX-mediated programmed cell death in Nicotiana benthamiana leaves. Characterization of isolate-specific effector candidates Iug6 and Iug9 and PaR candidate Iug18 revealed that they have a role in fungal propagation and pathogenicity. Moreover, Iug6 and Iug9 are located exclusively in the biotrophic interfacial complex (BIC) and their overexpression leads to suppression of defense-related gene expression in rice, suggesting that they might participate in biotrophy by inhibiting the SA and ET pathways within the host. Thus, our studies identify novel effector and PaR proteins involved in pathogenicity of the highly aggressive M. oryzae field isolate 98-06, and reveal molecular and genomic dynamics in the evolution of M. oryzae and rice host interactions.