Plant immunity suppression by an exo-ß-1,3-glucanase and an elongation factor 1α of the rice blast fungus.

Fungal cell walls undergo continual remodeling that generates ß-1,3-glucan fragments as products of endo-glycosyl hydrolases (GHs), which can be recognized as pathogen-associated molecular patterns (PAMPs) and trigger plant immune responses. How fungal pathogens suppress those responses is often poorly understood. Here, we study mechanisms underlying the suppression of ß-1,3-glucan-triggered plant immunity by the blast fungus Magnaporthe oryzae. We show that an exo-ß-1,3-glucanase of the GH17 family, named Ebg1, is important for fungal cell wall integrity and virulence of M. oryzae. Ebg1 can hydrolyze ß-1,3-glucan and laminarin into glucose, thus suppressing ß-1,3-glucan-triggered plant immunity. However, in addition, Ebg1 seems to act as a PAMP, independent of its hydrolase activity. This Ebg1-induced immunity appears to be dampened by the secretion of an elongation factor 1 alpha protein (EF1α), which interacts and co-localizes with Ebg1 in the apoplast. Future work is needed to understand the mechanisms behind Ebg1-induced immunity and its suppression by EF1α.

Identification and Characterization of Novel Candidate Effector Proteins from Magnaporthe oryzae.

The fungal pathogen Magnaporthe oryzae secretes a large number of effector proteins to facilitate infection, most of which are not functionally characterized. We selected potential candidate effector genes from the genome of M. oryzae, field isolate P131, and cloned 69 putative effector genes for functional screening. Utilizing a rice protoplast transient expression system, we identified that four candidate effector genes, GAS1, BAS2, MoCEP1 and MoCEP2 induced cell death in rice. In particular, MoCEP2 also induced cell death in Nicotiana benthamiana leaves through Agrobacteria-mediated transient gene expression. We further identified that six candidate effector genes, MoCEP3 to MoCEP8, suppress flg22-induced ROS burst in N. benthamiana leaves upon transient expression. These effector genes were highly expressed at a different stage after M. oryzae infection. We successfully knocked out five genes in M. oryzae, MoCEP1, MoCEP2, MoCEP3, MoCEP5 and MoCEP7. The virulence tests suggested that the deletion mutants of MoCEP2, MoCEP3 and MoCEP5 showed reduced virulence on rice and barley plants. Therefore, those genes play an important role in pathogenicity.

Magnaporthe oryzae endoplasmic reticulum membrane complex regulates the biogenesis of membrane proteins for pathogenicity.

In eukaryotes, the majority of newly synthesized integral membrane proteins are inserted into the endoplasmic reticulum (ER) membrane before transferred to their functional sites. The conserved ER membrane complex (EMC) takes part in the insertion process for tail-anchored membrane proteins. However, the function of EMC in phytopathogenic fungi has not been characterized. Here, we report the identification and functional characterization of two EMC subunits MoEmc5 and MoEmc2 in Magnaporthe oryzae. The knockout mutants ΔMoemc5 and ΔMoemc2 exhibit substantial defect in autophagy, pathogenicity, cell wall integrity, and magnesium ion sensitivity. We demonstrate that the autophagy process was severely impaired in the ΔMoemc5 and ΔMoemc2 mutants because of the low-protein steady-state level of Atg9, the sole membrane-associated autophagy protein. Furthermore, the protein level of membrane proteins Chs4, Fks1, and MoMnr2 is also significantly reduced in the ΔMoemc5 and ΔMoemc2 strains, leading to their supersensitivity to Calcofluor white, Congo red, and magnesium. In addition, MoEmc5, but not MoEmc2, acts as a magnesium transporter independent of its EMC function. Magnaporthe oryzae EMC regulates the biogenesis of membrane proteins for autophagy and virulence; therefore, EMC subunits could be potential targets for fungicide design in the future.

Two Species of Stemphylium, S. astragali and S. henanense sp. nov., Causing Leaf and Stem Spot Disease on Astragalus sinicus in China.

Astragalus sinicus is a versatile legume crop, primarily utilized as a green manure in China. During 2020 and 2021, A. sinicus plants exhibiting dark brown or reddish-brown lesions or spots on leaves and stems were collected from fields in the Henan, Sichuan, and Guangxi provinces of China. Sixteen single-spore isolates were isolated from the infected leaf and stem tissue samples. Phylogenetic analyses based on the concatenated internal transcribed spacer, gapdh, and cmdA sequences indicated that 14 of them belong to Stemphylium astragali, whereas two isolates can be well separated from other known species in this genus. Based on the morphological characteristics and nucleotide polymorphisms with sister taxa, the two isolates were identified as a new species named S. henanense. Furthermore, pathogenicity assays showed that the S. astragali and S. henanense isolates caused leaf and stem spot symptoms on A. sinicus. Altogether, we describe a new species of Stemphylium (i.e., S. henanense sp. nov.) causing leaf spot disease of A. sinicus. In addition, this is the first report of S. astragali causing stem spot disease of A. sinicus.

First Report of Leaf Spot Disease Caused by Boeremia linicola on Trifolium repens in China.

Trifolium repens L. (White clover) a multipurpose legume crop, primarily utilized as a green manure in China. In March 2018, field investigations showed that a leaf spot disease occurred on T. repens in three fields with 50% to 80% incidence (50 plants in each field were investigated) in Nanchong City, Sichuan Province of China. Infected leaves showed symptoms of irregular dark brown spots in the center of leaves or at the leaf margins. Symptomatic leaves were surface sterilized with 3% NaClO for 3 min followed by 75% ethanol for 30 s and then rinsed in sterile water three times. Thereafter, tissue samples from margins of individual lesions were placed on potato dextrose agar and incubated at 25°C in the dark. Four pure cultures (Y3-1; Y3-2; Y3-3; Y3-4) were obtained by single spore isolation. On oatmeal agar medium, a colony reached 57 mm diameter after 9 d in alternating light and dark at 25℃. Fimbriate aerial hypha were flat and compact with pale brown to dark brown color. Conidiogenous cells were hyaline, smooth, ampulliform to doliiform (n = 30), ranging from 4.1 to 10.5 µm long (6.6 ± 1.8 µm) × 2.2 to 5.7 µm wide (4.1 ± 0.8 µm). Conidia were ellipsoidal to cylindrical, hyaline, thin walled, smooth, aseptate, with 2 to 4 polar guttules (n = 50), ranging from 3.2 to 11.3 µm long (6.3 ± 1.1 µm) × 2.1 to 4.1 µm wide (2.8 ± 0.4 µm). Conidial matrix was whitish. Morphologically these isolates resembled species in Boeremia (Chen et al. 2015). Genomic DNA of each culture was extracted from mycelia using the quick and safe method (Chi et al. 2009). The 28S large subunit of nuclear ribosomal RNA (LSU) region, internal transcribed spacer (ITS) region, RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-α (TEF 1-α), ß-Tubulin (TUB2) were amplified with corresponding primers (Carbone and Kohn 1999; Liu et al. 1999; Rehner and Samuels 1994; Sung et al. 2007; Vilgalys and Hester 1990; White et al. 1990; Woudenberg et al.2009). Sequences were deposited in GenBank with accession numbers: ON705759 to ON705766, ON734043 to ON734046 and ON841585 to ON841592. Phylogenetic analysis was conducted with combined sequences of the five loci using the maximum likelihood (ML) and the maximum parsimony (MP) methods. The four isolates and the extype strain of B. linicola (CBS 116.76) clustered together with high bootstrap support (BS) values (MLBS = 100; MPBS = 98). All sequences showed 100% identity to those of CBS 116.76, except the ITS region of our isolates (ON705759 to ON705762), which show 99.6% identity to that of CBS 116.76. Based on morphological characteristics and phylogenetic results, our isolates were identified as B. linicola, although the morphological characteristics of CBS 116.76 had not been characterized. To assess pathogenicity, a conidial suspension (approximately 105 CFU/mL) of isolate Y3-1 was sprayed on 1-month-old healthy plants in a greenhouse at 22℃ to 28℃. Plants sprayed with sterilized water were used as negative controls. The test was conducted three times, each with 3 plants. After 10 days, the leaves of the plants showed irregular brown lesions that were similar to the symptoms observed in the field, control plants remained healthy. The pathogen was reisolated and confirmed to be B. linicola, thus completing the verification of Koch's Postulates. Compared to B. exigua, a causal pathogen associated with leaf spot on white clover reported by Wang et al (Wang et al. 2021), B. linicola produced larger conidia, and the two species did not cluster together in the phylogenetic tree. To our knowledge, this is the first report of leaf spot disease caused by B. linicola on Trifolium repens in China.

Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity.

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant-fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.

A cell death assay in barley and wheat protoplasts for identification and validation of matching pathogen AVR effector and plant NLR immune receptors.

BACKGROUND: Plant disease resistance to host-adapted pathogens is often mediated by host nucleotide-binding and leucine-rich repeat (NLR) receptors that detect matching pathogen avirulence effectors (AVR) inside plant cells. AVR-triggered NLR activation is typically associated with a rapid host cell death at sites of attempted infection and this response constitutes a widely used surrogate for NLR activation. However, it is challenging to assess this cell death in cereal hosts. RESULTS: Here we quantify cell death upon NLR-mediated recognition of fungal pathogen AVRs in mesophyll leaf protoplasts of barley and wheat. We provide measurements for the recognition of the fungal AVRs AvrSr50 and AVR a1 by their respective cereal NLRs Sr50 and Mla1 upon overexpression of the AVR and NLR pairs in mesophyll protoplast of both, wheat and barley. CONCLUSIONS: Our data demonstrate that the here described approach can be effectively used to detect and quantify death of wheat and barley cells induced by overexpression of NLR and AVR effectors or AVR effector candidate genes from diverse fungal pathogens within 24 h.

Multiple pairs of allelic MLA immune receptor-powdery mildew AVRA effectors argue for a direct recognition mechanism.

Nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins in plants and animals mediate intracellular pathogen sensing. Plant NLRs typically detect strain-specific pathogen effectors and trigger immune responses often linked to localized host cell death. The barley Mla disease resistance locus has undergone extensive functional diversification in the host population and encodes numerous allelic NLRs each detecting a matching isolate-specific avirulence effector (AVRA) of the fungal pathogen Blumeria graminis f. sp. hordei (Bgh). We report here the isolation of Bgh AVRa7, AVRa9, AVRa10, and AVRa22, which encode small secreted proteins recognized by allelic MLA7, MLA9, MLA10, and MLA22 receptors, respectively. These effectors are sequence-unrelated, except for allelic AVRa10 and AVRa22 that are co-maintained in pathogen populations in the form of a balanced polymorphism. Contrary to numerous examples of indirect recognition of bacterial effectors by plant NLRs, co-expression experiments with matching Mla-AVRa pairs indicate direct detection of the sequence-unrelated fungal effectors by MLA receptors.

Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen.

Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVRa gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVRa genes and identified AVRa1 and AVRa13, encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVRa1 and AVRa13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVRA1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVRA1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVRA1 Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation.

Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways.

Arabidopsis (Arabidopsis thaliana) penetration (PEN) genes quantitatively contribute to the execution of different forms of plant immunity upon challenge with diverse leaf pathogens. PEN3 encodes a plasma membrane-resident pleiotropic drug resistance-type ATP-binding cassette transporter and is thought to act in a pathogen-inducible and PEN2 myrosinase-dependent metabolic pathway in extracellular defense. This metabolic pathway directs the intracellular biosynthesis and activation of tryptophan-derived indole glucosinolates for subsequent PEN3-mediated efflux across the plasma membrane at pathogen contact sites. However, PEN3 also functions in abiotic stress responses to cadmium and indole-3-butyric acid (IBA)-mediated auxin homeostasis in roots, raising the possibility that PEN3 exports multiple functionally unrelated substrates. Here, we describe the isolation of a pen3 allele, designated pen3-5, that encodes a dysfunctional protein that accumulates in planta like wild-type PEN3. The specific mutation in pen3-5 uncouples PEN3 functions in IBA-stimulated root growth modulation, callose deposition induced with a conserved peptide epitope of bacterial flagellin (flg22), and pathogen-inducible salicylic acid accumulation from PEN3 activity in extracellular defense, indicating the engagement of multiple PEN3 substrates in different PEN3-dependent biological processes. We identified 4-O-ß-D-glucosyl-indol-3-yl formamide (4OGlcI3F) as a pathogen-inducible, tryptophan-derived compound that overaccumulates in pen3 leaf tissue and has biosynthesis that is dependent on an intact PEN2 metabolic pathway. We propose that a precursor of 4OGlcI3F is the PEN3 substrate in extracellular pathogen defense. These precursors, the shared indole core present in IBA and 4OGlcI3F, and allele-specific uncoupling of a subset of PEN3 functions suggest that PEN3 transports distinct indole-type metabolites in distinct biological processes.

The Arabidopsis PEPR pathway couples local and systemic plant immunity.

Recognition of microbial challenges leads to enhanced immunity at both the local and systemic levels. In Arabidopsis, EFR and PEPR1/PEPR2 act as the receptor for the bacterial elongation factor EF-Tu (elf18 epitope) and for the endogenous PROPEP-derived Pep epitopes, respectively. The PEPR pathway has been described to mediate defence signalling following microbial recognition. Here we show that PROPEP2/PROPEP3 induction upon pathogen challenges is robust against jasmonate, salicylate, or ethylene dysfunction. Comparative transcriptome profiling between Pep2- and elf18-treated plants points to co-activation of otherwise antagonistic jasmonate- and salicylate-mediated immune branches as a key output of PEPR signalling. Accordingly, as well as basal defences against hemibiotrophic pathogens, systemic immunity is reduced in pepr1 pepr2 plants. Remarkably, PROPEP2/PROPEP3 induction is essentially restricted to the pathogen challenge sites during pathogen-induced systemic immunity. Localized Pep application activates genetically separable jasmonate and salicylate branches in systemic leaves without significant PROPEP2/PROPEP3 induction. Our results suggest that local PEPR activation provides a critical step in connecting local to systemic immunity by reinforcing separate defence signalling pathways.

Structure and evolution of barley powdery mildew effector candidates.

BACKGROUND: Protein effectors of pathogenicity are instrumental in modulating host immunity and disease resistance. The powdery mildew pathogen of grasses Blumeria graminis causes one of the most important diseases of cereal crops. B. graminis is an obligate biotrophic pathogen and as such has an absolute requirement to suppress or avoid host immunity if it is to survive and cause disease. RESULTS: Here we characterise a superfamily predicted to be the full complement of Candidates for Secreted Effector Proteins (CSEPs) in the fungal barley powdery mildew parasite B. graminis f.sp. hordei. The 491 genes encoding these proteins constitute over 7% of this pathogen's annotated genes and most were grouped into 72 families of up to 59 members. They were predominantly expressed in the intracellular feeding structures called haustoria, and proteins specifically associated with the haustoria were identified by large-scale mass spectrometry-based proteomics. There are two major types of effector families: one comprises shorter proteins (100-150 amino acids), with a high relative expression level in the haustoria and evidence of extensive diversifying selection between paralogs; the second type consists of longer proteins (300-400 amino acids), with lower levels of differential expression and evidence of purifying selection between paralogs. An analysis of the predicted protein structures underscores their overall similarity to known fungal effectors, but also highlights unexpected structural affinities to ribonucleases throughout the entire effector super-family. Candidate effector genes belonging to the same family are loosely clustered in the genome and are associated with repetitive DNA derived from retro-transposons. CONCLUSIONS: We employed the full complement of genomic, transcriptomic and proteomic analyses as well as structural prediction methods to identify and characterize the members of the CSEPs superfamily in B. graminis f.sp. hordei. Based on relative intron position and the distribution of CSEPs with a ribonuclease-like domain in the phylogenetic tree we hypothesize that the associated genes originated from an ancestral gene, encoding a secreted ribonuclease, duplicated successively by repetitive DNA-driven processes and diversified during the evolution of the grass and cereal powdery mildew lineage.

Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism.

Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.

Uncoupling of sustained MAMP receptor signaling from early outputs in an Arabidopsis endoplasmic reticulum glucosidase II allele.

Recognition of microbe-associated molecular patterns (MAMPs), conserved structures typical of a microbial class, triggers immune responses in eukaryotes. This is accompanied by a diverse set of physiological responses that are thought to enhance defense activity in plants. However, the extent and mechanisms by which MAMP-induced events contribute to host immunity are poorly understood. Here we reveal Arabidopsis priority in sweet life4 (psl4) and psl5 mutants that are insensitive to the bacterial elongation factor (EF)-Tu epitope elf18 but responsive to flagellin epitope flg22. PSL4 and PSL5, respectively, identify beta- and alpha-subunits of endoplasmic reticulum-resident glucosidase II, which is essential for stable accumulation and quality control of the elf18 receptor EFR but not the flg22 receptor FLS2. We notice that EFR signaling is partially and differentially impaired without a significant decrease of the receptor steady-state levels in 2 weakly dysfunctional gIIalpha alleles, designated psl5-1 and rsw3. Remarkably, rsw3 plants exhibit marked supersusceptibility against a virulent bacterial phytopathogen despite nearly intact coactivation of MAPKs, reactive oxygen species, ethylene biosynthesis, and callose deposition in response to elf18, demonstrating that these signaling outputs alone are insufficient to mount effective immunity. However, rsw3 plants fail to maintain high transcript levels of defense-promoting WRKY, PR1, and PR2 genes at late time points (4 to 24 h) after elf18 elicitation. This points to an unexpected separation between initial and sustained activation of EFR-mediated signaling in the absence of proper glucosidase II-mediated endoplasmic reticulum quality control. Our findings strongly suggest the importance of sustained MAMP receptor signaling as a key step in the establishment of robust immunity.

Receptor quality control in the endoplasmic reticulum for plant innate immunity.

Pattern recognition receptors in eukaryotes initiate defence responses on detection of microbe-associated molecular patterns shared by many microbe species. The Leu-rich repeat receptor-like kinases FLS2 and EFR recognize the bacterial epitopes flg22 and elf18, derived from flagellin and elongation factor-Tu, respectively. We describe Arabidopsis 'priority in sweet life' (psl) mutants that show de-repressed anthocyanin accumulation in the presence of elf18. EFR accumulation and signalling, but not of FLS2, are impaired in psl1, psl2, and stt3a plants. PSL1 and PSL2, respectively, encode calreticulin3 (CRT3) and UDP-glucose:glycoprotein glycosyltransferase that act in concert with STT3A-containing oligosaccharyltransferase complex in an N-glycosylation pathway in the endoplasmic reticulum. However, EFR-signalling function is impaired in weak psl1 alleles despite its normal accumulation, thereby uncoupling EFR abundance control from quality control. Furthermore, salicylic acid-induced, but EFR-independent defence is weakened in psl2 and stt3a plants, indicating the existence of another client protein than EFR for this immune response. Our findings suggest a critical and selective function of N-glycosylation for different layers of plant immunity, likely through quality control of membrane-localized regulators.