Rab7/Retromer-based endolysosomal trafficking is essential for proper host invasion in rice blast.

Secretion is a fundamental process that plant pathogens utilize to deliver effectors into the host to downregulate immunity and promote infection. Here, we uncover a fascinating membrane trafficking and delivery route that originates from vacuolar membranes in Magnaporthe oryzae and conduits to the host interface and plasma membrane. To perform such secretory/trafficking function, MoRab7 first recruits the retromer complex to the vacuolar membrane, enabling recognition of a family of SNARE proteins, including MoSnc1. Live-cell imaging confirmed a highly dynamic vesicular trafficking of the retromer complex component(s) and MoSnc1 toward and across the host interface or plasma membrane, and subsequent fusion with target membranes. Interestingly, disruption of the MoRab7/Retromer/MoSnc1-based endolysosomal cascade affects effector secretion and fungal pathogenicity. Taken together, we discovered an unconventional protein and membrane trafficking route starting from the fungal endolysosomes to the M. oryzae-rice interaction interface and dissect the role of MoRab7/Retromer/MoSnc1 sorting machinery in effector secretion during biotrophy and invasive growth in rice blast fungus.

Subcellular compartmentation, interdependency and dynamics of the cyclic AMP-dependent PKA subunits during pathogenic differentiation in rice blast.

The cAMP-dependent PKA signalling plays a central role in growth, asexual development and pathogenesis in fungal pathogens. Here, we functionally characterised RPKA, the regulatory subunit of cAMP/PKA and studied the dynamics and organisation of the PKA subunits in the rice blast pathogen Magnaporthe oryzae. The RPKA subunit was essential for proper vegetative growth, asexual sporulation and surface hydrophobicity in M. oryzae. A spontaneous suppressor mutation, SMR19, that restored growth and conidiation in the RPKA deletion mutant was isolated and characterised. SMR19 enhanced conidiation and appressorium formation but failed to suppress the pathogenesis defects in rpkAΔ. The PKA activity was undetectable in the mycelial extracts of SMR19, which showed a single mutation (val242leu) in the highly conserved active site of the catalytic subunit (CPKA) of cAMP/PKA. The two subunits of cAMP/PKA showed different subcellular localisation patterns with RpkA being predominantly nucleocytoplasmic in conidia, while CpkA was largely cytosolic and/or vesicular. The CpkA anchored RpkA in cytoplasmic vesicles, and localisation of PKA in the cytoplasm was governed by CpkA in a cAMP-dependant or independent manner. We show that there exists a tight regulation of PKA subunits at the level of transcription, and the cAMP signalling is differentially compartmentalised in a stage-specific manner in rice blast.

The late endosomal HOPS complex anchors active G-protein signaling essential for pathogenesis in magnaporthe oryzae.

In Magnaporthe oryzae, the causal ascomycete of the devastating rice blast disease, the conidial germ tube tip must sense and respond to a wide array of requisite cues from the host in order to switch from polarized to isotropic growth, ultimately forming the dome-shaped infection cell known as the appressorium. Although the role for G-protein mediated Cyclic AMP signaling in appressorium formation was first identified almost two decades ago, little is known about the spatio-temporal dynamics of the cascade and how the signal is transmitted through the intracellular network during cell growth and morphogenesis. In this study, we demonstrate that the late endosomal compartments, comprising of a PI3P-rich (Phosphatidylinositol 3-phosphate) highly dynamic tubulo-vesicular network, scaffold active MagA/GαS, Rgs1 (a GAP for MagA), Adenylate cyclase and Pth11 (a non-canonical GPCR) in the likely absence of AKAP-like anchors during early pathogenic development in M. oryzae. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Remarkably, exogenous cAMP rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae. We propose that sequestration of key G-protein signaling components on dynamic late endosomes and/or endolysosomes, provides an effective molecular means to compartmentalize and control the spatio-temporal activation and rapid downregulation (likely via vacuolar degradation) of cAMP signaling amidst changing cellular geometry during pathogenic development in M. oryzae.

Draft genome sequence of Penicillium citrinum B9, a plant growth-promoting symbiont from barley rhizosphere.

Penicillium citrinum strain B9 is a plant growth-promoting fungus isolated from Barley (Hordeum vulgare) rhizosphere. We report the first draft genome of P. citrinum B9 assembled using single-molecule real-time sequencing and Illumina reads. The assembled genome spans 31.3 Mb comprising nine contigs and 10,106 protein-encoding genes.

Draft genome sequence of Xylaria bambusicola isolate GMP-LS, the root and basal stem rot pathogen of sugarcane in Indonesia.

The first draft genome of X. bambusicola GMP-LS, the causal pathogen of the Root, and Basal Stem Rot disease in Sugarcane is presented based on single-molecule real-time PacBio sequencing. Xylaria genome (72.43 Mb) is predicted to encode 13,430 proteins and will contribute to molecular understanding of fungal pathogenesis.

Functional analysis of auxin derived from a symbiotic mycobiont.

The biosynthesis of auxin or indole-3-acetic acid by microorganisms has a major impact on plant-microbe interactions. Several beneficial microbiota are known to produce auxin, which largely influences root development and growth in the host plants. Akin to findings in rhizobacteria, recent studies have confirmed the production of auxin by plant growth-promoting fungi too. Here, we show that Penicillium citrinum isolate B9 produces auxin as deduced by liquid chromatography tandem-mass spectrometry analysis. Such fungal auxin is secreted and contributes directly to enhanced root and shoot development and overall plant growth in Arabidopsis thaliana. Furthermore, auxin production by P. citrinum likely involves more than one tryptophan-dependent pathway. Using auxin biosynthesis inhibitor L-Kynurenine, we show that the indole-3-pyruvate pathway might be one of the key biosynthetic routes involved in such auxin production. Confocal microscopy of the DR5rev:GFP Arabidopsis reporter line helped demonstrate that P. citrunum B9-derived auxin is biologically active and is able to significantly enhance auxin signaling in roots during such improved root growth and plant development. Furthermore, the phenotypic growth defects arising from impaired auxin signaling in Arabidopsis taa1 mutant or upon L-Kynurenine treatment of wild-type Arabidopsis seedlings could be significantly alleviated by fungus B9-derived auxin, thus suggesting its positive role in plant growth promotion. Collectively, our results provide clear evidence that the production of auxin is one of the main mechanisms involved in induction of the beneficial plant growth by P. citrinum.

Penicillium citrinum Provides Transkingdom Growth Benefits in Choy Sum (Brassica rapa var. parachinensis).

Soil-borne beneficial microbes establish symbioses with plant hosts and play key roles during growth and development therein. In this study, two fungal strains, FLP7 and B9, were isolated from the rhizosphere microbiome associated with Choy Sum (Brassica rapa var. parachinensis) and barley (Hordeum vulgare), respectively. Sequence analyses of the internal transcribed spacer and 18S ribosomal RNA genes combined with colony and conidial morphology identified FLP7 and B9 to be Penicillium citrinum strains/isolates. Plant-fungus interaction assays revealed that isolate B9 showed significant growth promotion effects in Choy Sum plants cultivated in normal soil, as well as under phosphate-limiting conditions. In comparison to the mock control, B9-inoculated plants showed a 34% increase in growth in aerial parts and an 85% upsurge in the fresh weight of roots when cultivated in sterilized soil. The dry biomass of such fungus-inoculated Choy Sum increased by 39% and 74% for the shoots and roots, respectively. Root colonization assays showed that P. citrinum associates directly with the root surface but does not enter or invade the root cortex of the inoculated Choy Sum plants. Preliminary results also indicated that P. citrinum can promote growth in Choy Sum via volatile metabolites too. Interestingly, we detected relatively higher amounts of gibberellins and cytokinins in axenic P. citrinum culture filtrates through liquid chromatography-mass spectrometry analyses. This could plausibly explain the overall growth induction in P. citrinum-inoculated Choy Sum plants. Furthermore, the phenotypic growth defects associated with the Arabidopsis ga1 mutant could be chemically complemented by the exogenous application of P. citrinum culture filtrate, which also showed accumulation of fungus-derived active gibberellins. Our study underscores the importance of transkingdom beneficial effects of such mycobiome-assisted nutrient assimilation and beneficial fungus-derived phytohormone-like metabolites in the induction of robust growth in urban farmed crops.

Cpk2, a Catalytic Subunit of Cyclic AMP-PKA, Regulates Growth and Pathogenesis in Rice Blast.

The cAMP-Protein Kinase A signaling, anchored on CpkA, is necessary for appressorium development and host penetration, but indispensable for infectious growth in Magnaporthe oryzae. In this study, we identified and characterized the gene encoding the second catalytic subunit, CPK2, whose expression was found to be lower compared to CPKA at various stages of pathogenic growth in M. oryzae. Deletion of CPK2 caused no alterations in vegetative growth, conidiation, appressorium formation, or pathogenicity. Surprisingly, the cpkAΔcpk2Δ double deletion strain displayed significant reduction in growth rate and conidiation compared to the single deletion mutants. Interestingly, loss of CPKA and CPK2 resulted in morphogenetic defects in germ tubes (with curled/wavy and serpentine growth pattern) on hydrophobic surfaces, and a complete failure to produce appressoria therein, thus suggesting an important role for CPK2-mediated cAMP-PKA in surface sensing and response pathway. CPKA promoter-driven expression of CPK2 partially suppressed the defects in host penetration and pathogenicity in the cpkAΔ. Such ectopic CPK2 expressing strain successfully penetrated the rice leaves, but was unable to produce proper secondary invasive hyphae, thus underscoring the importance of CpkA in growth and differentiation in planta. The Cpk2-GFP localized to the nuclei and cytoplasmic vesicles in conidia and germ tubes. The Cpk2-GFP colocalized with CpkA-mCherry on vesicles in the cytosol, but such overlap was not evident in the nuclei. Our studies indicate that CpkA and Cpk2 share overlapping functions, but also play distinct roles during pathogenesis-associated signaling and morphogenesis in the rice blast fungus.