The potato rhizosphere microbiota correlated to the yield of three different regions in Korea.

We examined potato rhizosphere bacterial and fungal communities across three regions: Cheongju, Pyeongchang, and Gangneung. These regions have varying soil and climate conditions, resulting in different yields. We found that precipitation was the main limiting factor in our study while soil physiochemical factors affect bacterial and fungal microbiota in correlation with yield. Both bacterial and fungal microbiota showed distinct patterns according to the regions. ASVs positively correlated with yield were predominantly found in the Pyeongchang region which also produced the highest yields, while ASVs negatively correlated with yield were associated with Gangneung where the lowest yields were observed. The greatest bacterial and fungal diversity was detected in Pyeongchang consisting of Propionibacteriales, Burkholderiales, and Vicinamibacteriales. Gangneung, on the other hand primarily belong to Sordariales, Mortierellales, Cystofilobasidiales, and Tremellales. The putative yield-negative ASVs detected in Gangneung may have been influenced by drought stress. This work has highlighted key bacterial and fungal taxa as well as core taxa that may potentially be associated with high and low yields of potato in relation to metadata which includes soil chemical and physical parameters as well as weather data. Taken together we suggest that this information can be used to assess site suitability for potato production.

Attenuation of phytofungal pathogenicity of Ascomycota by autophagy modulators.

Autophagy in eukaryotes functions to maintain homeostasis by degradation and recycling of long-lived and unwanted cellular materials. Autophagy plays important roles in pathogenicity of various fungal pathogens, suggesting that autophagy is a novel target for development of antifungal compounds. Here, we describe bioluminescence resonance energy transfer (BRET)-based high-throughput screening (HTS) strategy to identify compounds that inhibit fungal ATG4 cysteine protease-mediated cleavage of ATG8 that is critical for autophagosome formation. We identified ebselen (EB) and its analogs ebselen oxide (EO) and 2-(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PT) as inhibitors of fungal pathogens Botrytis cinerea and Magnaporthe oryzae ATG4-mediated ATG8 processing. The EB and its analogs inhibit spore germination, hyphal development, and appressorium formation in Ascomycota pathogens, B. cinerea, M. oryzae, Sclerotinia sclerotiorum and Monilinia fructicola. Treatment with EB and its analogs significantly reduced fungal pathogenicity. Our findings provide molecular insights to develop the next generation of antifungal compounds by targeting autophagy in important fungal pathogens.

High-quality genome resource of a basidiomycetous yeast, Moesziomyces antarcticus isolate RS1, isolated from rice seed.

Moesziomyces antarcticus (anamorph: Pseudozyma antarctica) is a basidiomycetous yeast in the Ustilaginaceae family and is a core member of the rice seed microbiome. M. antarcticus RS1 was isolated from surface-sterilized rice seeds. This 18.287 Mb draft genome of M. antarcticus RS1 is comprised of a 60.8% GC content and 6,817 protein-coding genes.

Lyso-phosphatidylethanolamine triggers immunity against necrotrophs by promoting JA-signaling and ROS-homeostasis.

Modulation of the plant defense response by bioactive molecules is of increasing interest. However, despite plant cell lipids being one of the major cellular components, their role in plant immunity remains elusive. We found that the exogenous application of the cell-membrane localized phospholipid lyso-phosphatidylethanolamine (LPE) reprograms the plant transcript profile in favor of defense-associated genes thereby priming the plant immune system. Exogenous LPE application to different Arabidopsis accessions increases resistance against the necrotrophic pathogens, Botrytis cinerea and Cochliobolus heterostrophus. We found that the immunity-promoting effect of LPE is repealed in the jasmonic acid (JA) receptor mutant coi1, but multiplied in the JA-hypersensitive mutant feronia (fer-4). The JA-signaling repressor JAZ1 is degraded following LPE administration, suggesting that JA-signaling is promoted by LPE. Following LPE-treatment, reactive oxygen species (ROS) accumulation is affected in coi1 and fer-4. Moreover, FER signaling inhibitors of the RALF family are strongly expressed after LPE application, and RALF23 is internalized in stress granules, suggesting the LPE-mediated repression of FER-signaling by promoting RALF function. The in-situ increase of LPE-abundance in the LPE-catabolic mutants lpeat1 and lpeat2 elevates plant resistance to B. cinerea, in contrast to the endogenous LPE-deficient mutant pla2-alpha. We show that LPE increases plant resistance against necrotrophs by promoting JA-signaling and ROS-homeostasis, thereby paving the way for the LPE-targeted genomic engineering of crops to raise their ability to resist biotic threats.

Essential role of the CD docking motif of MPK4 in plant immunity, growth, and development.

MAPKs are universal eukaryotic signaling factors whose functioning is assumed to depend on the recognition of a common docking motif (CD) by its activators, substrates, and inactivators. We studied the role of the CD domain of Arabidopsis MPK4 by performing interaction studies and determining the ligand-bound MPK4 crystal structure. We revealed that the CD domain of MPK4 is essential for interaction and activation by its upstream MAPKKs MKK1, MKK2, and MKK6. Cys181 in the CD site of MPK4 was shown to become sulfenylated in response to reactive oxygen species in vitro. To test the function of C181 in vivo, we generated wild-type (WT) MPK4-C181, nonsulfenylatable MPK4-C181S, and potentially sulfenylation mimicking MPK4-C181D lines in the mpk4 knockout background. We analyzed the phenotypes in growth, development, and stress responses, revealing that MPK4-C181S has WT activity and complements the mpk4 phenotype. By contrast, MPK4-C181D cannot be activated by upstream MAPKK and cannot complement the phenotypes of mpk4. Our findings show that the CD motif is essential and is required for activation by upstream MAPKK for MPK4 function. Furthermore, growth, development, or immunity functions require upstream activation of the MPK4 protein kinase.

Plant domestication shapes rhizosphere microbiome assembly and metabolic functions.

BACKGROUND: The rhizosphere microbiome, which is shaped by host genotypes, root exudates, and plant domestication, is crucial for sustaining agricultural plant growth. Despite its importance, how plant domestication builds up specific rhizosphere microbiomes and metabolic functions, as well as the importance of these affected rhizobiomes and relevant root exudates in maintaining plant growth, is not well understood. Here, we firstly investigated the rhizosphere bacterial and fungal communities of domestication and wild accessions of tetraploid wheat using amplicon sequencing (16S and ITS) after 9 years of domestication process at the main production sites in China. We then explored the ecological roles of root exudation in shaping rhizosphere microbiome functions by integrating metagenomics and metabolic genomics approaches. Furthermore, we established evident linkages between root morphology traits and keystone taxa based on microbial culture and plant inoculation experiments. RESULTS: Our results suggested that plant rhizosphere microbiomes were co-shaped by both host genotypes and domestication status. The wheat genomes contributed more variation in the microbial diversity and composition of rhizosphere bacterial communities than fungal communities, whereas plant domestication status exerted much stronger influences on the fungal communities. In terms of microbial interkingdom association networks, domestication destabilized microbial network and depleted the abundance of keystone fungal taxa. Moreover, we found that domestication shifted the rhizosphere microbiome from slow growing and fungi dominated to fast growing and bacteria dominated, thereby resulting in a shift from fungi-dominated membership with enrichment of carbon fixation genes to bacteria-dominated membership with enrichment of carbon degradation genes. Metagenomics analyses further indicated that wild cultivars of wheat possess higher microbial function diversity than domesticated cultivars. Notably, we found that wild cultivar is able to harness rhizosphere microorganism carrying N transformation (i.e., nitrification, denitrification) and P mineralization pathway, whereas rhizobiomes carrying inorganic N fixation, organic N ammonification, and inorganic P solubilization genes are recruited by the releasing of root exudates from domesticated wheat. More importantly, our metabolite-wide association study indicated that the contrasting functional roles of root exudates and the harnessed keystone microbial taxa with different nutrient acquisition strategies jointly determined the aboveground plant phenotypes. Furthermore, we observed that although domesticated and wild wheats recruited distinct microbial taxa and relevant functions, domestication-induced recruitment of keystone taxa led to a consistent growth regulation of root regardless of wheat domestication status. CONCLUSIONS: Our results indicate that plant domestication profoundly influences rhizosphere microbiome assembly and metabolic functions and provide evidence that host plants are able to harness a differentiated ecological role of root-associated keystone microbiomes through the release of root exudates to sustain belowground multi-nutrient cycles and plant growth. These findings provide valuable insights into the mechanisms underlying plant-microbiome interactions and how to harness the rhizosphere microbiome for crop improvement in sustainable agriculture. Video Abstract.

Nuclear effectors of plant pathogens: Distinct strategies to be one step ahead.

Nuclear effector proteins released by bacteria, oomycete, nematode, and fungi burden the global environment and crop yield. Microbial effectors are key weapons in the evolutionary arms race between plants and pathogens, vital in determining the success of pathogenic colonization. Secreted effectors undermine a multitude of host cellular processes depending on their target destination. Effectors are classified by their localization as either extracellular (apoplastic) or intracellular. Intracellular effectors can be further subclassified by their compartment such as the nucleus, cytoplasm or chloroplast. Nuclear effectors bring into question the role of the plant nucleus' intrinsic defence strategies and their vulnerability to effector-based manipulation. Nuclear effectors interfere with multiple nuclear processes including the epigenetic regulation of the host chromatin, the impairment of the trans-kingdom antifungal RNAi machinery, and diverse classes of immunity-associated host proteins. These effector-targeted pathways are widely conserved among different hosts and regulate a broad array of plant cellular processes. Thus, these nuclear sites constitute meaningful targets for effectors to subvert the plant defence system and acquire resources for pathogenic propagation. This review provides an extensive and comparative compilation of diverse plant microbe nuclear effector libraries, thereby highlighting the distinct and conserved mechanisms these effectors employ to modulate plant cellular processes for the pathogen's profit.

The nuclear effector MoHTR3 of Magnaporthe oryzae modulates host defence signalling in the biotrophic stage of rice infection.

Fungal effectors play a pivotal role in suppressing the host defence system, and their evolution is highly dynamic. By comparative sequence analysis of plant-pathogenic fungi and Magnaporthe oryzae, we identified the small secreted C2 H2 zinc finger protein MoHTR3. MoHTR3 exhibited high conservation in M. oryzae strains but low conservation among other plant-pathogenic fungi, suggesting an emerging evolutionary selection process. MoHTR3 is exclusively expressed in the biotrophic stage of fungal invasion, and the encoded protein localizes to the biotrophic interfacial complex (BIC) and the host cell nucleus. The signal peptide crucial for MoHTR3' secretion to the BIC and the protein section required for its translocation to the nucleus were both identified by a functional protein domain study. The host-nuclear localization of MoHTR3 suggests a function as a transcriptional modulator of host defence gene induction. After ΔMohtr3 infection, the expression of jasmonic acid- and ethylene-associated genes was diminished in rice, in contrast to when the MoHTR3-overexpressing strain (MoHTR3ox) was applied. The transcript levels of salicylic acid- and defence-related genes were also affected after ΔMohtr3 and MoHTR3ox application. In pathogenicity assays, ΔMohtr3 was indistinguishable from the wild type. However, MoHTR3ox-infected plants showed diminished lesion formation and hydrogen peroxide accumulation, accompanied by a decrease in susceptibility, suggesting that the MoHTR3-induced manipulation of host cells affects host-pathogen interaction. MoHTR3 emphasizes the role of the host nucleus as a critical target for the pathogen-driven manipulation of host defence mechanisms and underscores the ongoing evolution of rice blast's arms race.

Dysbiotic but nonpathogenic shift in the fecal mycobiota of patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is closely associated with the oral and gut microbiomes. Fungal cell wall components initiate inflammatory arthritis in mouse models. However, little is known regarding the role of the fungal community in the pathogenesis of RA. To evaluate the association between RA and the gut microbiome, investigations of bacterial and fungal communities in patients with RA are necessary. Therefore, we investigated the compositions and associations of fecal bacterial and fungal communities in 30 healthy controls and 99 patients with RA. The relative abundances of Bifidobacterium and Blautia decreased, whereas the relative abundance of Streptococcus increased, in patients with RA. The relative abundance of Candida in the fecal fungal community was higher in patients with RA than in healthy controls, while the relative abundance of Aspergillus was higher in healthy controls than in patients with RA. Candida species-specific gene amplification showed that C. albicans was the most abundant species of Candida. Ordination analysis and random forest classification models supported the findings of structural changes in bacterial and fungal communities. Aspergillus was the core fecal fungal genus in healthy controls, although Saccharomyces spp. are typically predominant in Western cohorts. In addition, bacterial-fungal association analyses showed that the hub node had shifted from fungi to bacteria in patients with RA. The finding of fungal dysbiosis in patients with RA suggests that fungi play critical roles in the fecal microbial communities and pathogenesis of RA.

Comparative profiling of canonical and non-canonical small RNAs in the rice blast fungus, Magnaporthe oryzae.

RNA interference (RNAi) is divided into canonical, Dicer-dependent and non-canonical, Dicer-independent pathways according to Dicer protein dependency. However, sRNAs processed in a Dicer-independent manner have not been reported in plant pathogenic fungi, including Magnaporthe oryzae. We comparatively profiled the Dicer-dependent and -independent sRNAs of M. oryzae. Dicer-dependent sRNAs were 19-24-nt in length, had low strand-specificity, and showed a preference for uracil at the 5'-end. By contrast, Dicer-independent sRNAs presented irregular patterns in length distribution, high strand-specificity, and a preference for cytosine at the penultimate position. Dicer-dependent sRNA loci were mainly associated with LTR-transposons, while Dicer-independent sRNAs were associated with protein-coding genes and transposons. We identified MoERI-1, a non-canonical RNAi component, and profiled the sRNA and mRNA transcriptomes of ΔMoeri-1 at the mycelia and conidiation stages, as the mutant showed increased conidiation. We found that genes involved in conidiation and cell cycle were upregulated by MoERI-1 deletion. Furthermore, a comparison between sRNA and mRNA transcriptome revealed that MoERI-1-dependent sRNAs mediate the regulation of gene expression. Overall, these results showed that M. oryzae has non-canonical RNAi pathways distinct to the Dicer-dependent manner and exploits MoERI-1-dependent sRNAs to regulate the conidiation process.

Cross-kingdom co-occurrence networks in the plant microbiome: Importance and ecological interpretations.

Microbial co-occurrence network analysis is being widely used for data exploration in plant microbiome research. Still, challenges lie in how well these microbial networks represent natural microbial communities and how well we can interpret and extract eco-evolutionary insights from the networks. Although many technical solutions have been proposed, in this perspective, we touch on the grave problem of kingdom-level bias in network representation and interpretation. We underscore the eco-evolutionary significance of using cross-kingdom (bacterial-fungal) co-occurrence networks to increase the network's representability of natural communities. To do so, we demonstrate how ecosystem-level interpretation of plant microbiome evolution changes with and without multi-kingdom analysis. Then, to overcome oversimplified interpretation of the networks stemming from the stereotypical dichotomy between bacteria and fungi, we recommend three avenues for ecological interpretation: (1) understanding dynamics and mechanisms of co-occurrence networks through generalized Lotka-Volterra and consumer-resource models, (2) finding alternative ecological explanations for individual negative and positive fungal-bacterial edges, and (3) connecting cross-kingdom networks to abiotic and biotic (host) environments.

Ambivalent response in pathogen defense: A double-edged sword?

Plants possess effective immune systems that defend against most microbial attackers. Recent plant immunity research has focused on the classic binary defense model involving the pivotal role of small-molecule hormones in regulating the plant defense signaling network. Although most of our current understanding comes from studies that relied on information derived from a limited number of pathosystems, newer studies concerning the incredibly diverse interactions between plants and microbes are providing additional insights into other novel mechanisms. Here, we review the roles of both classical and more recently identified components of defense signaling pathways and stress hormones in regulating the ambivalence effect during responses to diverse pathogens. Because of their different lifestyles, effective defense against biotrophic pathogens normally leads to increased susceptibility to necrotrophs, and vice versa. Given these opposing forces, the plant potentially faces a trade-off when it mounts resistance to a specific pathogen, a phenomenon referred to here as the ambivalence effect. We also highlight a novel mechanism by which translational control of the proteins involved in the ambivalence effect can be used to engineer durable and broad-spectrum disease resistance, regardless of the lifestyle of the invading pathogen.

Longitudinal transmission of bacterial and fungal communities from seed to seed in rice.

Vertical transmission of microbes is crucial for the persistence of host-associated microbial communities. Although vertical transmission of seed microbes has been reported from diverse plants, ecological mechanisms and dynamics of microbial communities from parent to progeny remain scarce. Here we reveal the veiled ecological mechanism governing transmission of bacterial and fungal communities in rice across two consecutive seasons. We identify 29 bacterial and 34 fungal members transmitted across generations. Abundance-based regression models allow to classify colonization types of the microbes. We find that they are late colonizers dominating each community at the ripening stage. Ecological models further show that the observed temporal colonization patterns are affected by niche change and neutrality. Source-sink modeling reveals that parental seeds and stem endosphere are major origins of progeny seed microbial communities. This study gives empirical evidence for ecological mechanism and dynamics of bacterial and fungal communities as an ecological continuum during seed-to-seed transmission.

A comparative genomic analysis of lichen-forming fungi reveals new insights into fungal lifestyles.

Lichen-forming fungi are mutualistic symbionts of green algae or cyanobacteria. We report the comparative analysis of six genomes of lichen-forming fungi in classes Eurotiomycetes and Lecanoromycetes to identify genomic information related to their symbiotic lifestyle. The lichen-forming fungi exhibited genome reduction via the loss of dispensable genes encoding plant-cell-wall-degrading enzymes, sugar transporters, and transcription factors. The loss of these genes reflects the symbiotic biology of lichens, such as the absence of pectin in the algal cell wall and obtaining specific sugars from photosynthetic partners. The lichens also gained many lineage- and species-specific genes, including those encoding small secreted proteins. These genes are primarily induced during the early stage of lichen symbiosis, indicating their significant roles in the establishment of lichen symbiosis.Our findings provide comprehensive genomic information for six lichen-forming fungi and novel insights into lichen biology and the evolution of symbiosis.

Protocol on the fabrication of monocrystalline thin semiconductor via crack-assisted layer exfoliation technique for photoelectrochemical water-splitting.

Thin semiconductors attract huge interest due to their cost-effective, flexible, lightweight, and semi-transparent properties. Here, we present a protocol on the preparation of thin semiconductor via controlled crack-assisted layer exfoliation technique. The protocol details the fabrication procedure for producing thin monocrystalline semiconductors with thicknesses in the range of a few tens of micrometers from thick donor substrates. In addition, we describe proof-of-concept application of the thin semiconductors for photoelectrochemical water-splitting to produce hydrogen fuel. For complete details on the use and execution of this protocol, please refer to Lee et al. (2021).

ROS homeostasis mediated by MPK4 and SUMM2 determines synergid cell death.

Sexual plant reproduction depends on the attraction of sperm-cell delivering pollen tubes (PT) by two synergids, followed by their programmed cell death (PCD) in Arabidopsis. Disruption of the mitogen-activated protein kinase 4 (MPK4) by pathogenic effectors activates the resistance protein (R) SUMM2-mediated immunity and cell death. Here we show that synergid preservation and reactive oxygen species (ROS) homeostasis are intimately linked and maintained by MPK4. In mpk4, ROS levels are increased and synergids prematurely undergo PCD before PT-reception. However, ROS scavengers and the disruption of SUMM2, in mpk4, restore ROS homeostasis, synergid maintenance and PT perception, demonstrating that the guardian of MPK4, SUMM2, triggers synergid-PCD. In mpk4/summ2, PTs show a feronia-like overgrowth phenotype. Our results show that immunity-associated PCD and synergid cell death during plant reproduction are regulated by MPK4 underscoring an underlying molecular mechanism for the suppression of plant reproduction during systemic R-mediated immunity.

Alternative splicing diversifies the transcriptome and proteome of the rice blast fungus during host infection.

Alternative splicing (AS) contributes to diversifying and regulating cellular responses to environmental conditions and developmental cues by differentially producing multiple mRNA and protein isoforms from a single gene. Previous studies on AS in pathogenic fungi focused on profiling AS isoforms under a limited number of conditions. We analysed AS profiles in the rice blast fungus Magnaporthe oryzae, a global threat to rice production, using high-quality transcriptome data representing its vegetative growth (mycelia) and multiple host infection stages. We identified 4,270 AS isoforms derived from 2,413 genes, including 499 genes presumably regulated by infection-specific AS. AS appears to increase during infection, with 32.7% of the AS isoforms being produced during infection but absent in mycelia. Analysis of the isoforms observed at each infection stage showed that 636 AS isoforms were more abundant than corresponding annotated mRNAs, especially after initial hyphal penetration into host cell. Many such dominant isoforms were predicted to encode regulatory proteins such as transcription factors and phospho-transferases. We also identified the genes encoding distinct proteins via AS and confirmed the translation of some isoforms via a proteomic analysis, suggesting potential AS-mediated neo-functionalization of some genes during infection. Comprehensive profiling of the pattern of genome-wide AS during multiple stages of rice-M. oryzae interaction established a foundational resource that will help investigate the role and regulation of AS during rice infection.

Genome-wide profiling of long non-coding RNA of the rice blast fungus Magnaporthe oryzae during infection.

BACKGROUND: Long non-coding RNAs (lncRNAs) play essential roles in developmental processes and disease development at the transcriptional and post-transcriptional levels across diverse taxa. However, only few studies have profiled fungal lncRNAs in a genome-wide manner during host infection. RESULTS: Infection-associated lncRNAs were identified using lncRNA profiling over six stages of host infection (e.g., vegetative growth, pre-penetration, biotrophic, and necrotrophic stages) in the model pathogenic fungus, Magnaporthe oryzae. We identified 2,601 novel lncRNAs, including 1,286 antisense lncRNAs and 980 intergenic lncRNAs. Among the identified lncRNAs, 755 were expressed in a stage-specific manner and 560 were infection-specifically expressed lncRNAs (ISELs). To decipher the potential roles of lncRNAs during infection, we identified 365 protein-coding genes that were associated with 214 ISELs. Analysis of the predicted functions of these associated genes suggested that lncRNAs regulate pathogenesis-related genes, including xylanases and effectors. CONCLUSIONS: The ISELs and their associated genes provide a comprehensive view of lncRNAs during fungal pathogen-plant interactions. This study expands new insights into the role of lncRNAs in the rice blast fungus, as well as other plant pathogenic fungi.

A rice gene encoding glycosyl hydrolase plays contrasting roles in immunity depending on the type of pathogens.

Because pathogens use diverse infection strategies, plants cannot use one-size-fits-all defence and modulate defence responses based on the nature of pathogens and pathogenicity mechanism. Here, we report that a rice glycoside hydrolase (GH) plays contrasting roles in defence depending on whether a pathogen is hemibiotrophic or necrotrophic. The Arabidopsis thaliana MORE1 (Magnaporthe oryzae resistance 1) gene, encoding a member of the GH10 family, is needed for resistance against M. oryzae and Alternaria brassicicola, a fungal pathogen infecting A. thaliana as a necrotroph. Among 13 rice genes homologous to MORE1, 11 genes were induced during the biotrophic or necrotrophic stage of infection by M. oryzae. CRISPR/Cas9-assisted disruption of one of them (OsMORE1a) enhanced resistance against hemibiotrophic pathogens M. oryzae and Xanthomonas oryzae pv. oryzae but increased susceptibility to Cochliobolus miyabeanus, a necrotrophic fungus, suggesting that OsMORE1a acts as a double-edged sword depending on the mode of infection (hemibiotrophic vs. necrotrophic). We characterized molecular and cellular changes caused by the loss of MORE1 and OsMORE1a to understand how these genes participate in modulating defence responses. Although the underlying mechanism of action remains unknown, both genes appear to affect the expression of many defence-related genes. Expression patterns of the GH10 family genes in A. thaliana and rice suggest that other members also participate in pathogen defence.