The phosphorylation landscape of infection-related development by the rice blast fungus.

Many of the world's most devastating crop diseases are caused by fungal pathogens that elaborate specialized infection structures to invade plant tissue. Here, we present a quantitative mass-spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins following germination on a hydrophobic surface, revealing major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring (PRM) to identify phosphoproteins regulated by the fungal Pmk1 MAPK that controls plant infection by M. oryzae. We define 32 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of regulator Vts1 is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for the control of plant diseases.

RRM Transcription Factors Interact with NLRs and Regulate Broad-Spectrum Blast Resistance in Rice.

Nucleotide-binding site leucine-rich repeat (NLR) receptors perceive pathogen effectors and trigger plant immunity. However, the mechanisms underlying NLR-triggered defense responses remain obscure. The recently discovered Pigm locus in rice encodes a cluster of NLRs, including PigmR, which confers broad-spectrum resistance to blast fungus. Here, we identify PIBP1 (PigmR-INTERACTING and BLAST RESISTANCE PROTEIN 1), an RRM (RNA-recognition motif) protein that specifically interacts with PigmR and other similar NLRs to trigger blast resistance. PigmR-promoted nuclear accumulation of PIBP1 ensures full blast resistance. We find that PIBP1 and a homolog, Os06 g02240, bind DNA and function as unconventional transcription factors at the promoters of the defense genes OsWAK14 and OsPAL1, activating their expression. Knockout of PIBP1 and Os06 g02240 greatly attenuated blast resistance. Collectively, our study discovers previously unappreciated RRM transcription factors that directly interact with NLRs to activate plant defense, establishing a direct link between transcriptional activation of immune responses with NLR-mediated pathogen perception.

Characterization of a unique polysaccharide monooxygenase from the plant pathogen Magnaporthe oryzae.

Blast disease in cereal plants is caused by the fungus Magnaporthe oryzae and accounts for a significant loss in food crops. At the outset of infection, expression of a putative polysaccharide monooxygenase (MoPMO9A) is increased. MoPMO9A contains a catalytic domain predicted to act on cellulose and a carbohydrate-binding domain that binds chitin. A sequence similarity network of the MoPMO9A family AA9 showed that 220 of the 223 sequences in the MoPMO9A-containing cluster of sequences have a conserved unannotated region with no assigned function. Expression and purification of the full length and two MoPMO9A truncations, one containing the catalytic domain and the domain of unknown function (DUF) and one with only the catalytic domain, were carried out. In contrast to other AA9 polysaccharide monooxygenases (PMOs), MoPMO9A is not active on cellulose but showed activity on cereal-derived mixed (1→3, 1→4)-ß-D-glucans (MBG). Moreover, the DUF is required for activity. MoPMO9A exhibits activity consistent with C4 oxidation of the polysaccharide and can utilize either oxygen or hydrogen peroxide as a cosubstrate. It contains a predicted 3-dimensional fold characteristic of other PMOs. The DUF is predicted to form a coiled-coil with six absolutely conserved cysteines acting as a zipper between the two α-helices. MoPMO9A substrate specificity and domain architecture are different from previously characterized AA9 PMOs. The results, including a gene ontology analysis, support a role for MoPMO9A in MBG degradation during plant infection. Consistent with this analysis, deletion of MoPMO9A results in reduced pathogenicity.

The RLCK subfamily VII-4 controls pattern-triggered immunity and basal resistance to bacterial and fungal pathogens in rice.

Receptor-like cytoplasmic kinases (RLCKs) mediate the intracellular signaling downstream of pattern-recognition receptors (PRRs). Several RLCKs from subfamily VII of rice (Oryza sativa) have important roles in plant immunity, but the role of RLCK VII-4 in pattern-triggered immune (PTI) signaling and resistance to pathogens has not yet been investigated. Here, we generated by multiplex clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated genome editing rice sextuple mutant lines where the entire RLCK VII-4 subfamily is inactivated and then analyzed the resulting lines for their response to chitin and flg22 and for their immunity to Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae. Analysis of the rlckvii-4 mutants revealed that they have an impaired reactive oxygen system burst and reduced defense gene expression in response to flg22 and chitin. This indicates that members of the rice RLCK VII-4 subfamily are required for immune signaling downstream of multiple PRRs. Furthermore, we found that the rice RLCK VII-4 subfamily is important for chitin-induced callose deposition and mitogen-activated protein kinase activation and that it is crucial for basal resistance against Xoo and M. oryzae pathogens. This establishes that the RLCK VII-4 subfamily has critical functions in the regulation of multiple PTI pathways in rice and opens the way for deciphering the precise role of its members in the control of rice PTI.

Mitochondrial Outer Membrane Translocase MoTom20 Modulates Mitochondrial Morphology and Is Important for Infectious Growth of the Rice Blast Fungus Magnaporthe oryzae.

Mitochondria are highly dynamic organelles that constantly change their morphology to adapt to the cellular environment through fission and fusion, which is critical for a cell to maintain normal cellular functions. Despite the significance of this process in the development and pathogenicity of the rice blast fungus Magnaporthe oryzae, the underlying mechanism remains largely elusive. Here, we identified and characterized a mitochondrial outer membrane translocase, MoTom20, in M. oryzae. Targeted gene deletion revealed that MoTom20 plays an important role in vegetative growth, conidiogenesis, penetration, and infectious growth of M. oryzae. The growth rate, conidial production, appressorium turgor, and pathogenicity are decreased in the ΔMotom20 mutant compared with the wild-type and complemented strains. Further analysis revealed that MoTom20 localizes in mitochondrion and plays a key role in regulating mitochondrial fission and fusion balance, which is critical for infectious growth. Finally, we found that MoTom20 is involved in fatty-acid utilization, and its yeast homolog ScTom20 is able to rescue the defects of ΔMotom20 in mitochondrial morphology and pathogenicity. Overall, our data demonstrate that MoTom20 is a key regulator for mitochondrial morphology maintenance, which is important for infectious growth of the rice blast fungus M. oryzae. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Paths of Least Resistance: Unconventional Effector Secretion by Fungal and Oomycete Plant Pathogens.

Effector secretion by different routes mediates the molecular interplay between host plant and pathogen, but mechanistic details in eukaryotes are sparse. This may limit the discovery of new effectors that could be utilized for improving host plant disease resistance. In fungi and oomycetes, apoplastic effectors are secreted via the conventional endoplasmic reticulum (ER)-Golgi pathway, while cytoplasmic effectors are packaged into vesicles that bypass Golgi in an unconventional protein secretion (UPS) pathway. In Magnaporthe oryzae, the Golgi bypass UPS pathway incorporates components of the exocyst complex and a t-SNARE, presumably to fuse Golgi bypass vesicles to the fungal plasma membrane. Upstream, cytoplasmic effector mRNA translation in M. oryzae requires the efficient decoding of AA-ending codons. This involves the modification of wobble uridines in the anticodon loop of cognate tRNAs and fine-tunes cytoplasmic effector translation and secretion rates to maintain biotrophic interfacial complex integrity and permit host infection. Thus, plant-fungal interface integrity is intimately tied to effector codon usage, which is a surprising constraint on pathogenicity. Here, we discuss these findings within the context of fungal and oomycete effector discovery, delivery, and function in host cells. We show how cracking the codon code for unconventional cytoplasmic effector secretion in M. oryzae has revealed AA-ending codon usage bias in cytoplasmic effector mRNAs across kingdoms, including within the RxLR-dEER motif-encoding sequence of a bona fide Phytophthora infestans cytoplasmic effector, suggesting its subjection to translational speed control. By focusing on recent developments in understanding unconventional effector secretion, we draw attention to this important but understudied area of host-pathogen interactions. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Silencing Osa-miR827 via CRISPR/Cas9 protects rice against the blast fungus Magnaporthe oryzae.

MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression at the post-transcriptional level. In plants, miRNAs participate in diverse developmental processes and adaptive responses to biotic and abiotic stress. MiR827 has long been recognized to be involved in plant responses to phosphate starvation. In rice, the miR827 regulates the expression of OsSPX-MFS1 and OsSPX-MFS2, these genes encoding vacuolar phosphate transporters. In this study, we demonstrated that miR827 plays a role in resistance to infection by the fungus Magnaporthe oryzae in rice. We show that MIR827 overexpression enhances susceptibility to infection by M. oryzae which is associated to a weaker induction of defense gene expression during pathogen infection. Conversely, CRISPR/Cas9-induced mutations in the MIR827 gene completely abolish miR827 production and confer resistance to M. oryzae infection. This resistance is accompanied by a reduction of leaf Pi content compared to wild-type plants, whereas Pi levels increase in leaves of the blast-susceptible miR827 overexpressor plants. In wild-type plants, miR827 accumulation in leaves decreases during the biotrophic phase of the infection process. Taken together, our data indicates that silencing MIR827 confers resistance to M. oryzae infection in rice while further supporting interconnections between Pi signaling and immune signaling in plants. Unravelling the role of miR827 during M. oryzae infection provides knowledge to improve blast resistance in rice by CRISPR/Cas9-editing of MIR827.

Sirt5-mediated lysine desuccinylation regulates oxidative stress adaptation in Magnaporthe oryzae during host intracellular infection.

Plant pathogenic fungi elaborate numerous detoxification strategies to suppress host reactive oxygen species (ROS), but their coordination is not well-understood. Here, we show that Sirt5-mediated protein desuccinylation in Magnaporthe oryzae is central to host ROS detoxification. SIRT5 encodes a desuccinylase important for virulence via adaptation to host oxidative stress. Quantitative proteomics analysis identified a large number of succinylated proteins targeted by Sirt5, most of which were mitochondrial proteins involved in oxidative phosphorylation, TCA cycle, and fatty acid oxidation. Deletion of SIRT5 resulted in hypersuccinylation of detoxification-related enzymes, and significant reduction in NADPH : NADP+ and GSH : GSSG ratios, disrupting redox balance and impeding invasive growth. Sirt5 desuccinylated thioredoxin Trx2 and glutathione peroxidase Hyr1 to activate their enzyme activity, likely by affecting proper folding. Altogether, this work demonstrates the importance of Sirt5-mediated desuccinylation in controlling fungal process required for detoxifying host ROS during M. oryzae infection.

Vacuolar recruitment of retromer by a SNARE complex enables infection-related trafficking in rice blast.

The retromer complex is a conserved sorting machinery that maintains cellular protein homeostasis by transporting vesicles containing cargo proteins to defined destinations. It is known to sort proteins at the vacuole membranes for retrograde trafficking, preventing their degradation in the vacuole. However, the detailed mechanism of retromer recruitment to the vacuole membrane has not yet been elucidated. Here, we show that the vacuolar SNARE complex MoPep12-MoVti1-MoVam7-MoYkt6 regulates retromer-mediated vesicle trafficking by recruiting the retromer to the vacuole membrane, which promotes host invasion in Magnaporthe oryzae. Such recruitment is also essential for the retrieval of the autophagy regulator MoAtg8 and enables appressorium-mediated host penetration. Furthermore, the vacuolar SNARE subunits are involved in suppressing the host defense response by regulating the deployment of retromer-MoSnc1-mediated effector secretion. Altogether, our results provide insights into the mechanism of vacuolar SNAREs-dependent retromer recruitment which is necessary for pathogenicity-related membrane trafficking events in the rice blast fungus.

Effector-triggered susceptibility by the rice blast fungus Magnaporthe oryzae.

Rice blast, the most destructive disease of cultivated rice world-wide, is caused by the filamentous fungus Magnaporthe oryzae. To cause disease in plants, M. oryzae secretes a diverse range of effector proteins to suppress plant defense responses, modulate cellular processes, and support pathogen growth. Some effectors can be secreted by appressoria even before host penetration, while others accumulate in the apoplast, or enter living plant cells where they target specific plant subcellular compartments. During plant infection, the blast fungus induces the formation of a specialized plant structure known as the biotrophic interfacial complex (BIC), which appears to be crucial for effector delivery into plant cells. Here, we review recent advances in the cell biology of M. oryzae-host interactions and show how new breakthroughs in disease control have stemmed from an increased understanding of effector proteins of M. oryzae are deployed and delivered into plant cells to enable pathogen invasion and host susceptibility.

Strigolactone deficiency induces jasmonate, sugar and flavonoid phytoalexin accumulation enhancing rice defense against the blast fungus Pyricularia oryzae.

Strigolactones (SLs) are carotenoid-derived phytohormones that regulate plant growth and development. While root-secreted SLs are well-known to facilitate plant symbiosis with beneficial microbes, the role of SLs in plant interactions with pathogenic microbes remains largely unexplored. Using genetic and biochemical approaches, we demonstrate a negative role of SLs in rice (Oryza sativa) defense against the blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae). We found that SL biosynthesis and perception mutants, and wild-type (WT) plants after chemical inhibition of SLs, were less susceptible to P. oryzae. Strigolactone deficiency also resulted in a higher accumulation of jasmonates, soluble sugars and flavonoid phytoalexins in rice leaves. Likewise, in response to P. oryzae infection, SL signaling was downregulated, while jasmonate and sugar content increased markedly. The jar1 mutant unable to synthesize jasmonoyl-l-isoleucine, and the coi1-18 RNAi line perturbed in jasmonate signaling, both accumulated lower levels of sugars. However, when WT seedlings were sprayed with glucose or sucrose, jasmonate accumulation increased, suggesting a reciprocal positive interplay between jasmonates and sugars. Finally, we showed that functional jasmonate signaling is necessary for SL deficiency to induce rice defense against P. oryzae. We conclude that a reduction in rice SL content reduces P. oryzae susceptibility by activating jasmonate and sugar signaling pathways, and flavonoid phytoalexin accumulation.

OsEIL2 balances rice immune responses against (hemi)biotrophic and necrotrophic pathogens via the salicylic acid and jasmonic acid synergism.

Plants typically activate distinct defense pathways against various pathogens. Heightened resistance to one pathogen often coincides with increased susceptibility to another pathogen. However, the underlying molecular basis of this antagonistic response remains unclear. Here, we demonstrate that mutants defective in the transcription factor ETHYLENE-INSENSITIVE 3-LIKE 2 (OsEIL2) exhibited enhanced resistance to the biotrophic bacterial pathogen Xanthomonas oryzae pv oryzae and to the hemibiotrophic fungal pathogen Magnaporthe oryzae, but enhanced susceptibility to the necrotrophic fungal pathogen Rhizoctonia solani. Furthermore, necrotroph-induced OsEIL2 binds to the promoter of OsWRKY67 with high affinity, leading to the upregulation of salicylic acid (SA)/jasmonic acid (JA) pathway genes and increased SA/JA levels, ultimately resulting in enhanced resistance. However, biotroph- and hemibiotroph-induced OsEIL2 targets OsERF083, resulting in the inhibition of SA/JA pathway genes and decreased SA/JA levels, ultimately leading to reduced resistance. Our findings unveil a previously uncharacterized defense mechanism wherein two distinct transcriptional regulatory modules differentially mediate immunity against pathogens with different lifestyles through the transcriptional reprogramming of phytohormone pathway genes.

Phenazine biosynthesis protein MoPhzF regulates appressorium formation and host infection through canonical metabolic and noncanonical signaling function in Magnaporthe oryzae.

Microbe-produced secondary metabolite phenazine-1-carboxylic acid (PCA) facilitates pathogen virulence and defense mechanisms against competitors. Magnaporthe oryzae, a causal agent of the devastating rice blast disease, needs to compete with other phyllosphere microbes and overcome host immunity for successful colonization and infection. However, whether M. oryzae produces PCA or it has any other functions remains unknown. Here, we found that the MoPHZF gene encodes the phenazine biosynthesis protein MoPhzF, synthesizes PCA in M. oryzae, and regulates appressorium formation and host virulence. MoPhzF is likely acquired through an ancient horizontal gene transfer event and has a canonical function in PCA synthesis. In addition, we found that PCA has a role in suppressing the accumulation of host-derived reactive oxygen species (ROS) during infection. Further examination indicated that MoPhzF recruits both the endoplasmic reticulum membrane protein MoEmc2 and the regulator of G-protein signaling MoRgs1 to the plasma membrane (PM) for MoRgs1 phosphorylation, which is a critical regulatory mechanism in appressorium formation and pathogenicity. Collectively, our studies unveiled a canonical function of MoPhzF in PCA synthesis and a noncanonical signaling function in promoting appressorium formation and host infection.

A distinct protein posttranslational modifications-linked OsATL32-OsPPKL2-OsGSK2 loop modulates rice immunity against blast disease.

Protein posttranslational modifications play crucial roles in plant immunity through modulating a complicated signaling network mediated by different hormones. We previously demonstrated that OsATL32, an ATL-type E3 ligase, negatively contributes to rice immunity against Magnaporthe oryzae. Here, we show that OsATL32 forms a loop with OsPPKL2 and OsGSK2 through distinct protein posttranslational modifications to modulate rice immunity. OsATL32 ubiquitinates OsPPKL2, a protein phosphatase with Kelch-like repeat domains that exerts positive roles in regulating rice immunity against M. oryzae and chitin-triggered immune responses, for degradation. The glycogen synthase kinase 2 (OsGSK2), which acts as a negative regulator of rice immunity against M. oryzae and chitin-triggered immune responses, phosphorylates OsATL32 to elevate its protein stability and E3 ligase activity on OsPPKL2. Moreover, OsPPKL2 directly dephosphorylates OsGSK2, affecting its kinase activity on substrates including OsATL32 for phosphorylation. Like OsGSK2 as a BR signaling repressor, OsATL32 negatively regulates BR signaling; conversely, OsPPKL2 plays a positive role in BR signaling. These findings provide a molecular mechanism in which OsATL32 serves as a node connecting BR signaling and immunity by associating with OsPPKL2 and OsGSK2, assembling into a distinct protein posttranslational modifications-linked loop that functions in rice BR signaling and immunity.

Proteomic signatures uncover phenotypic plasticity of susceptible and resistant genotypes by wall remodelers in rice blast.

Molecular communication between macromolecules dictates extracellular matrix (ECM) dynamics during pathogen recognition and disease development. Extensive research has shed light on how plant immune components are activated, regulated and function in response to pathogen attack. However, two key questions remain largely unresolved: (i) how does ECM dynamics govern susceptibility and disease resistance, (ii) what are the components that underpin these phenomena? Rice blast, caused by Magnaporthe oryzae adversely affects rice productivity. To understand ECM regulated genotype-phenotype plasticity in blast disease, we temporally profiled two contrasting rice genotypes in disease and immune state. Morpho-histological, biochemical and electron microscopy analyses revealed that increased necrotic lesions accompanied by electrolyte leakage governs disease state. Wall carbohydrate quantification showed changes in pectin level was more significant in blast susceptible compared to blast resistant cultivar. Temporally resolved quantitative disease- and immune-responsive ECM proteomes identified 308 and 334 proteins, respectively involved in wall remodelling and integrity, signalling and disease/immune response. Pairwise comparisons between time and treatment, messenger ribonucleic acid expression, diseasome and immunome networks revealed novel blast-related functional modules. Data demonstrated accumulation of α-galactosidase and phosphatase were associated with disease state, while reactive oxygen species, induction of Lysin motif proteins, CAZymes and extracellular Ca-receptor protein govern immune state.

OsHRZ1 negatively regulates rice resistant to Magnaporthe oryzae infection by targeting OsVOZ2.

Rice blast disease caused by Magnaporthe oryzae significantly reduces yield production. Blast resistance is closely associated with iron (Fe) status, but the mechanistic basis linking iron status to immune function in rice remains largely unknown. Here, iron-binding haemerythrin RING ubiquitin ligases OsHRZ1 was confirmed to play key roles in iron-mediated rice blast resistance. The expression of OsHRZ1 was suppressed by M. oryzae inoculation and high iron treatment. Both mutants of OsHRZ1 enhanced rice resistance to M. oryzae. OsPR1a was up-regulated in OsHRZ1 mutants. Yeast two-hybrid, bimolecular fluorescence complementation, and Co-IP assay results indicated that OsHRZ1 interacts with Vascular Plant One Zinc Finger 2 (OsVOZ2) in the nucleus. Additionally, the vitro ubiquitination assay indicated that OsHRZ1 can ubiquitinate OsVOZ2 and mediate the degradation of OsVOZ2. The mutants of OsVOZ2 showed reduced resistance to M. oryzae and down-regulated the expression of OsPR1a. Yeast one-hybrid, EMSA, and dual-luciferase reporter assay results indicated that OsVOZ2 directly binds to the promoter of OsPR1a, activating its expression. In summary, OsHRZ1 plays an important role in rice disease resistance by mediated degradation of OsVOZ2 thus shaping PR gene expression dynamics in rice cells. This highlights an important link between iron signaling and rice pathogen defenses.

Dihydroorotase MoPyr4 is required for development, pathogenicity, and autophagy in rice blast fungus.

Dihydroorotase (DHOase) is the third enzyme in the six enzymatic reaction steps of the endogenous pyrimidine nucleotide de novo biosynthesis pathway, which is a metabolic pathway conserved in both bacteria and eukaryotes. However, research on the biological function of DHOase in plant pathogenic fungi is very limited. In this study, we identified and named MoPyr4, a homologous protein of Saccharomyces cerevisiae DHOase Ura4, in the rice blast fungus Magnaporthe oryzae and investigated its ability to regulate fungal growth, pathogenicity, and autophagy. Deletion of MoPYR4 led to defects in growth, conidiation, appressorium formation, the transfer and degradation of glycogen and lipid droplets, appressorium turgor accumulation, and invasive hypha expansion in M. oryzae, which eventually resulted in weakened fungal pathogenicity. Long-term replenishment of exogenous uridine-5'-phosphate (UMP) can effectively restore the phenotype and virulence of the ΔMopyr4 mutant. Further study revealed that MoPyr4 also participated in the regulation of the Pmk1-MAPK signaling pathway, co-localized with peroxisomes for the oxidative stress response, and was involved in the regulation of the Osm1-MAPK signaling pathway in response to hyperosmotic stress. In addition, MoPyr4 interacted with MoAtg5, the core protein involved in autophagy, and positively regulated autophagic degradation. Taken together, our results suggested that MoPyr4 for UMP biosynthesis was crucial for the development and pathogenicity of M. oryzae. We also revealed that MoPyr4 played an essential role in the external stress response and pathogenic mechanism through participation in the Pmk1-MAPK signaling pathway, peroxisome-related oxidative stress response mechanism, the Osm1-MAPK signaling pathway and the autophagy pathway.

Allelic variation in rice blast resistance: a pathway to sustainable disease management.

Rice blast is a major problem in agriculture, affecting rice production and threatening food security worldwide. This disease, caused by the fungus Magnaporthe oryzae, has led to a lot of research since the discovery of the first resistance gene, pib, in 1999. Researchers have now identified more than 50 resistance genes on eight of the twelve chromosomes in rice, each targeting different strains of the pathogen.These genes are spread out across seventeen different loci. These genes, which primarily code for nucleotide-binding and leucine-rich repeat proteins, play an important part in the defense of rice against the pathogen, either alone or in combination with other genes. An important characteristic of these genes is the allelic or paralogous interactions that exist within these loci. These relationships contribute to the gene's increased capacity for evolutionary adaptation. The ability of resistance proteins to recognize and react to novel effectors is improved by the frequent occurrence of variations within the domains that are responsible for recognizing pathogen effectors. The purpose of this review is to summarize the progress that has been made in identifying these essential genes and to investigate the possibility of utilizing the allelic variants obtained from these genes in future rice breeding efforts to increase resistance to rice blast.

Antifungal characterizations of a novel endo-ß-1,6-glucanase from Flavobacterium sp. NAU1659.

ß-1,6-Glucan plays a crucial role in fungal cell walls by linking the outer layer of mannoproteins and the inner layer of ß-1,3-glucan, contributing significantly to the maintenance of cell wall rigidity. Therefore, the hydrolysis of ß-1,6-glucan by ß-1,6-glucanase directly leads to the disintegration of the fungal cell wall. Here, a novel ß-1,6-glucanase FlGlu30 was identified from the endophytic Flavobacterium sp. NAU1659 and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction conditions of purified FlGlu30 were 50℃ and pH 6.0, resulting in a specific activity of 173.1 U/mg using pustulan as the substrate. The hydrolyzed products of FlGlu30 to pustulan were mainly gentianose within 1 h of reaction. With the extension of reaction time, gentianose was gradually hydrolyzed to glucose, indicating that FlGlu30 is an endo-ß-1,6-glucanase. The germination of Magnaporthe oryzae Guy11 spores could not be inhibited by FlGlu30, but the appressorium formation of spores was completely inhibited under the concentration of 250.0 U/mL FlGlu30. The disruptions of cell wall and accumulation of intracellular reactive oxide species (ROS) were observed in FlGlu30-treated M. oryzae Guy11 cells, suggesting the significant importance of ß-1,6-glucan as a potential antifungal target and the potential application of FlGlu30. KEY POINTS: • ß-1,6-Glucan is a key component maintaining the rigid structure of fungal cell wall. • ß-1,6-Glucanase is an antifungal protein with significant potential applications. • FlGlu30 is the first reported ß-1, 6-glucanase derived from Flavobacterium.

A Reevaluation of Phylogenomic Data Reveals that Current Understanding in Wheat Blast Population Biology and Epidemiology Is Obfuscated by Oversights in Population Sampling.

Wheat blast, caused by the Pyricularia oryzae Triticum lineage (PoT), first emerged in Brazil and quickly spread to neighboring countries. Its recent appearance in Bangladesh and Zambia highlights a need to understand the disease's population biology and epidemiology so as to mitigate pandemic outbreaks. Current knowledge is mostly based on characterizations of Brazilian wheat blast isolates and comparison with isolates from non-wheat, endemic grasses. These foregoing studies concluded that the wheat blast population lacks host specificity and, as a result, undergoes extensive gene flow with populations infecting non-wheat hosts. Additionally, based on genetic similarity between wheat blast and isolates infecting Urochloa species, it was proposed that the disease originally emerged via a host jump from this grass and that Urochloa likely plays a central role in wheat blast epidemiology owing to its widespread use as a pasture grass. However, due to inconsistencies with broader phylogenetic studies, we suspected that these seminal studies had not actually sampled the populations normally found on endemic grasses and, instead, had repeatedly isolated members of PoT and the related Lolium pathogen lineage (PoL1). Re-analysis of the Brazilian data as part of a comprehensive, global, phylogenomic dataset that included a small number of South American isolates sampled away from wheat confirmed our suspicion and identified four new P. oryzae lineages on grass hosts. As a result, the conclusions underpinning current understanding in wheat blast's evolution, population biology, and epidemiology are unsubstantiated and could be equivocal.