Increasing evidence suggests that the endophytic fungus Piriformospora indica helps plants overcome various abiotic stresses, especially heavy metals. However, the mechanism of heavy metal tolerance has not yet been elucidated. Here, the role of P. indica in alleviating cadmium (Cd) toxicities in tobacco was investigated. It was found that P. indica improved Cd tolerance to tobacco, increasing Cd accumulation in roots but decreasing Cd accumulation in leaves. The colonization of P. indica altered the subcellular repartition of Cd, increasing the Cd proportion in cell walls while reducing the Cd proportion in membrane/organelle and soluble fractions. During Cd stress, P. indica significantly enhanced the peroxidase (POD) activity and glutathione (GSH) content in tobacco. The spatial distribution of GSH was further visualized by Raman spectroscopy, showing that GSH was distributed in the cortex of P. indica-inoculated roots while in the epidermis of the control roots. A LC-MS/MS-based label-free quantitative technique evaluated the differential proteomics of P. indica treatment vs. control plants under Cd stress. The expressions of peroxidase, glutathione synthase, and photosynthesis-related proteins were significantly upregulated. This study provided extensive evidence for how P. indica enhances Cd tolerance in tobacco at physiological, cytological, and protein levels.

Endophytic fungi are essential microorganisms in promoting plant health. However, the mechanism of endophytic fungi regulating root microbiota to enhance crop production and resistance remains unclear. We aimed i) to explore the microbial alteration driven by endophytic Acrocalymma vagum in developing crop yield and rice resistance; ii) to reveal the mechanism of root-released compound stimulated by A. vagum in recruiting benefit microbes. The microbiome was applied in a culture-dependent and culture-independent method to study the microbial communities of the A. vagum-rice holobiont using 16S rRNA and ITS gene metabarcoding. Non-target metabolome identified distinct metabolites responsible for community variations. Label-free proteomic analyses investigated the association between primary genes related to the holobiont formation. CRISPR/Cas9 technique and homologous recombination replacement were used to characterize the functions of putative genes. A. vagum enhanced cultivated rice yield by 5.73 ± 1.76 % and induced 83.24 ± 9.86 % control efficiency against rice blast. We discovered that A. vagum simplified rice microbial structure based on co-occurrence networks, by lowering the proportion of potentially pathogenic predominant Burkholderia and driving rice to recruit beneficial Lactobacillus, Sarocladium and Nigrospora to promote rice growth with the increases of 44.41 ± 5.10 % shoot height and 70.21 ± 9.57 % shoot biomass. Moreover, the holobiont released coumaric and trans-ferulic acids to attract beneficial microbes. 206 rice proteins were notably up-regulated in the holobiont, particularly the OsPrxs. CRISPR/Cas9-edited mutants of OsPRX70 and OsPRX95 reduced the promotion effect of A. vagum on rice growth. Furthermore, the pathways of 39 overexpressed proteins in A. vagum were enriched in invading the host and inducing resistance. The knockouts of AvGH3, AvGH7, AvMFS1, and AvCBA transformed A. vagum role from endophyte to pathogen. The A. vagum-rice holobiont releases recruitment signals and improves the rice community structure. We provide ecological and molecular evidence to confirm the mutualism of endophyte-plant-promoting growth and disease resistance.

Magnaporthe oryzae (synonym Pyricularia oryzae) is a filamentous fungal pathogen that causes major yield losses in cultivated rice worldwide. However, the mechanisms of infection of M. oryzae are not well characterized. The VPS13 proteins play vital roles in various biological processes in many eukaryotic organisms, including in the organization of actin cytoskeleton, vesicle trafficking, mitochondrial fusion, and phagocytosis. Nevertheless, the function of the Vps13 protein in plant pathogenic fungi has not been explored. Here, we analysed the biological functions of the Vps13 protein in the development and pathogenicity of M. oryzae. Deletion mutants of MoVps13 significantly reduced the conidiation and decreased the rate of fungal infection on hosts. Moreover, the loss of MoVps13 resulted in defective cell wall integrity (CWI) and plasma membrane (PM) homeostasis when treated with chemicals for inducing cell wall stress (200 mg/mL Congo Red or 0.005% SDS) and sphingolipid synthesis inhibitors (2 μM myriocin or 2 μM amphotericin B). This indicated that MoVps13 is also involved in cell wall synthesis and sphingolipid synthesis. Through immunoblotting, autophagic flux detection, co-localization, and chemical drug sensitivity assays, we confirmed the involvement of Movps13 in ER-phagy and the response to ER stress. Additionally, we generated the C-terminal structure of MoVps13 with high accuracy using the alphaflod2 database. Our experimental evidence indicates that MoVps13 is an important virulence factor that regulates the pathogenicity of M. oryzae by controlling CWI, lipid metabolism and the ER-phagy pathway. These results have expanded our knowledge about pathogenic fungi and will help exploration for novel therapeutic strategies against the rice blast fungus.

Mildew damage is a major reason for chestnut poor quality and yield loss. In this study, a near-infrared hyperspectral imaging system in the 874–1734 nm spectral range was applied to detect the mildew damage to chestnuts caused by blue mold. Principal component analysis (PCA) scored images were firstly employed to qualitatively and intuitively distinguish moldy chestnuts from healthy chestnuts. Spectral data were extracted from the hyperspectral images. A successive projections algorithm (SPA) was used to select 12 optimal wavelengths. Artificial neural networks, including back propagation neural network (BPNN), evolutionary neural network (ENN), extreme learning machine (ELM), general regression neural network (GRNN) and radial basis neural network (RBNN) were used to build models using the full spectra and optimal wavelengths to distinguish moldy chestnuts. BPNN and ENN models using full spectra and optimal wavelengths obtained satisfactory performances, with classification accuracies all surpassing 99%. The results indicate the potential for the rapid and non-destructive detection of moldy chestnuts by hyperspectral imaging, which would help to develop online detection system for healthy and blue mold infected chestnuts.

Wild rice (Oryza granulata) is a natural resource pool containing abundant unknown endophytic fungi species. There are few reports on the endophytic fungi in wild rice. Here, one isolate recovered from wild rice roots was identified as a new speciesPseudophialophora oryzaesp. nov based on the molecular phylogeny and morphological characteristics. Fluorescent protein-expressingP. oryzaewas used to monitor the fungal colonization pattern. Hyphae invaded the epidermis to the inner cortex but not into the root stele. The inoculation ofP. oryzaepromoted the rice growth, with the growth parameters of chlorophyll content, shoot height, root length, fresh shoot weight, fresh root weight and dry weight increasing by 24.10, 35.32, 19.35, 90.00, 33.3, and 79.17%, respectively.P. oryzaeinduced up-regulation of nitrate transporterOsPTR9and potassium transporterOsHAK16by 7.28 ± 0.84 and 2.57 ± 0.80 folds, promoting nitrogen and potassium elements absorption. In addition,P. oryzaealso conferred a systemic resistance against rice blast, showing a 72.65 and 75.63% control rate in sterile plates and potting conditions. This systemic resistance was mediated by the strongly up-regulated expression of resistance-related genesNAC,OsSAUR2,OsWRKY71,EL5, andPR1α. SinceP. oryzaecan promote rice growth, biomass and induce systemic disease resistance, it can be further developed as a new biogenic agent for agricultural production, providing a new approach for biocontrol of rice blast.

Microbes employ effectors to disrupt immune responses and promote host colonization. Conserved motifs including RXLR, LFLAK-HVLVxxP (CRN), Y/F/WxC, CFEM, LysM, Chitin-bind, DPBB_1 (PNPi), and Cutinase have been discovered to play crucial roles in the functioning of effectors in filamentous fungi. Nevertheless, little is known about effectors with conserved motifs in endophytes. This research aims to discover the effector genes with conserved motifs in the genome of rice endophyte Falciphora oryzae. SignalP identified a total of 622 secreted proteins, out of which 227 were predicted as effector candidates by EffectorP. By utilizing HMM features, we discovered a total of 169 effector candidates with conserved motifs and three novel motifs. Effector candidates containing LysM, CFEM, DPBB_1, Cutinase, and Chitin_bind domains were conserved across species. In the transient expression assay, it was observed that one CFEM and one LysM activated cell death in tobacco leaves. Moreover, two CFEM and one Chitin_bind inhibited cell death induced by Bax protein. At various points during the infection, the genes’ expression levels were increased. These results will help to identify functional effector proteins involving omics methods using new bioinformatics tools, thus providing a basis for the study of symbiosis mechanisms.