The structural repertoire of Fusarium oxysporum f. sp. lycopersici effectors revealed by experimental and computational studies.

Plant pathogens secrete proteins, known as effectors, that function in the apoplast or inside plant cells to promote virulence. Effector recognition by cell-surface or cytosolic receptors results in the activation of defence pathways and plant immunity. Despite their importance, our general understanding of fungal effector function and recognition by immunity receptors remains poor. One complication often associated with effectors is their high sequence diversity and lack of identifiable sequence motifs precluding prediction of structure or function. In recent years, several studies have demonstrated that fungal effectors can be grouped into structural classes, despite significant sequence variation and existence across taxonomic groups. Using protein X-ray crystallography, we identify a new structural class of effectors hidden within the secreted in xylem (SIX) effectors from Fusarium oxysporum f. sp. lycopersici (Fol). The recognised effectors Avr1 (SIX4) and Avr3 (SIX1) represent the founding members of the Fol dual-domain (FOLD) effector class, with members containing two distinct domains. Using AlphaFold2, we predicted the full SIX effector repertoire of Fol and show that SIX6 and SIX13 are also FOLD effectors, which we validated experimentally for SIX6. Based on structural prediction and comparisons, we show that FOLD effectors are present within three divisions of fungi and are expanded in pathogens and symbionts. Further structural comparisons demonstrate that Fol secretes effectors that adopt a limited number of structural folds during infection of tomato. This analysis also revealed a structural relationship between transcriptionally co-regulated effector pairs. We make use of the Avr1 structure to understand its recognition by the I receptor, which leads to disease resistance in tomato. This study represents an important advance in our understanding of Fol-tomato, and by extension plant-fungal interactions, which will assist in the development of novel control and engineering strategies to combat plant pathogens.

Mutation in Polycomb repressive complex 2 gene OsFIE2 promotes asexual embryo formation in rice.

Prevention of autonomous division of the egg apparatus and central cell in a female gametophyte before fertilization ensures successful reproduction in flowering plants. Here we show that rice ovules of Polycomb repressive complex 2 (PRC2) Osfie1 and Osfie2 double mutants exhibit asexual embryo and autonomous endosperm formation at a high frequency, while ovules of single Osfie2 mutants display asexual pre-embryo-like structures at a lower frequency without fertilization. Earlier onset, higher penetrance and better development of asexual embryos in the double mutants compared with those in Osfie2 suggest that the autonomous endosperm facilitated asexual embryo development. Transcriptomic analysis showed that male genome-expressed OsBBM1 and OsWOX8/9 were activated in the asexual embryos. Similarly, the maternal alleles of the paternally expressed imprinted genes were activated in the autonomous endosperm, suggesting that the egg apparatus and central cell convergently adopt PRC2 to maintain the non-dividing state before fertilization, possibly through silencing of the maternal alleles of male genome-expressed genes.

A chitosan-coated lentinan-loaded calcium alginate hydrogel induces broad-spectrum resistance to plant viruses by activating Nicotiana benthamiana calmodulin-like (CML) protein 3.

Control of plant virus diseases largely depends on the induced plant defence achieved by the external application of synthetic chemical inducers with the ability to modify defence-signalling pathways. However, most of the molecular mechanisms underlying these chemical inducers remain unknown. Here, we developed a chitosan-coated lentinan-loaded hydrogel and discovered how it protects plants from different virus infections. The hydrogel was synthesized by coating chitosan on the surface of the calcium alginate-lentinan (LNT) hydrogel (SL-gel) to form a CSL-gel. CSL-gels exhibit the capacity to prolong the stable release of lentinan and promote Ca2+ release. Application of CSL-gels on the root of plants induces broad-spectrum resistance against plant viruses (TMV, TRV, PVX and TuMV). RNA-seq analysis identified that Nicotiana benthamiana calmodulin-like protein gene 3 (NbCML3) is upregulated by the sustained release of Ca2+ from the CSL-gel, and silencing and overexpression of NbCML alter the susceptibility and resistance of tobacco to TMV. Our findings provide evidence that this novel and synthetic CSL-gel strongly inhibits the infection of plant viruses by the sustainable release of LNT and Ca2+ . This study uncovers a novel mode of action by which CSL-gels trigger NbCML3 expression through the stable and sustained release of Ca2+ .

Cell-free protein synthesis of CD1E and B2M protein and in vitro interaction.

CD1E, one of the most important glycolipid antigens on T cell membranes, is required for glycolipid antigen presentation on the cell surface. Cell-based recombinant expression systems have many limitations for synthesizing transmembrane proteins such as CD1E, including low protein yields and miss folding. To overcome these challenges, here we successfully synthesized high-quality soluble CD1E using an E.coli cell-free protein synthesis system (CFPS) with the aid of detergent. Following purification by Ni2+ affinity chromatography, we were able to obtain CD1E with ≥90% purity. Furthermore, we used the string website to predict the protein interaction network of CD1E and identified a potential binding partner━B2M. Similarly, we synthesized soluble B2M in the E.coli CFPS. Finally, we verified the interaction between CD1E and B2M by using Surface Plasmon Resonance (SPR). Taken together, the methods described here provide an alternative way to obtain active transmembrane protein and may facilitate future structural and functional studies on CD1E.

Detection and Quantification of Anthracnose Pathogen Colletotrichum fructicola in Cultivated Tea-Oil Camellia Species from Southern China Using a DNA-Based qPCR Assay.

Tea-oil Camellia species as edible-oil producing trees are widely cultivated in southern China. Camellia anthracnose that is mainly caused by Colletotrichum fructicola is a major disease of tea-oil trees. However, rapid detection and precise quantification of C. fructicola in different Camellia species that are crucial for the fundamental study of this pathosystem and effective disease management remain largely unexplored. Here, we developed a sensitive, rapid, and accurate method for quantifying C. fructicola growth in different Camellia species using a quantitative PCR assay. Amplified C. fructicola DNA using ITS-specific primers is relatively compared with the amplification of Camellia oleifera using the TUB gene. We determined that the fungal growth is tightly associated with the disease development in Ca. oleifera following C. fructicola infection in a time-course manner. This assay is highly sensitive, as fungal growth was detected in six different inoculated tea-oil Camellia species without visible disease lesion symptoms. Additionally, this method was validated by quantifying the Camellia anthracnose in orchards that did not show any disease symptoms. This assay enables the rapid, highly sensitive, and precise detection and quantification of C. fructicola growth in different tea-oil Camellia species, which will have a practical application for early diagnosis of anthracnose disease under asymptomatic conditions in Camellia breeding and field and will facilitate the development of tea-oil trees and C. fructicola interaction as a mold system to study woody plant and fungal pathogens interaction.

Mutation in a chlorophyll-binding motif of Brassica ferrochelatase enhances both heme and chlorophyll biosynthesis.

The heme branch of tetrapyrrole biosynthesis contributes to the regulation of chlorophyll levels. However, the mechanism underlying the balance between chlorophyll and heme synthesis remains elusive. Here, we identify a dark green leaf mutant, dg, from an ethyl methanesulfonate (EMS)-induced mutant library of Chinese cabbage. The dg phenotype is caused by an amino acid substitution in the conserved chlorophyll a/b-binding motif (CAB) of ferrochelatase 2 (BrFC2). This mutation increases the formation of BrFC2 homodimer to promote heme production. Moreover, wild-type BrFC2 and dBrFC2 interact with protochlorophyllide (Pchlide) oxidoreductase B1 and B2 (BrPORB1 and BrPORB2), and dBrFC2 exhibits higher binding ability to substrate Pchlide, thereby promoting BrPORBs-catalyzed production of chlorophyllide (Chlide), which can be directly converted into chlorophyll. Our results show that dBrFC2 is a gain-of-function mutation contributing to balancing heme and chlorophyll synthesis via a regulatory mechanism in which dBrFC2 promotes BrPORB enzymatic reaction to enhance chlorophyll synthesis.

Metabolomic and transcriptomic analyses identify quinic acid protecting eggplant from damage caused by western flower thrips.

BACKGROUND: Western flower thrips are considered the major insect pest of horticultural crops worldwide, causing economic and yield loss to Solanaceae crops. The eggplant (Solanum melongena L.) resistance against thrips remains largely unexplored. This work aims to identify thrips-resistant eggplants and dissect the molecular mechanisms underlying this resistance using the integrated metabolomic and transcriptomic analyses of thrips-resistant and -susceptible cultivars. RESULTS: We developed a micro-cage thrips bioassay to identify thrips-resistant eggplant cultivars, and highly resistant cultivars were identified from wild eggplant relatives. Metabolomic profiles of thrips-resistant and -susceptible eggplant were compared using the gas chromatography-mass spectrometry (GC-MS)-based approach, resulting in the identification of a higher amount of quinic acid in thrips-resistant eggplant compared to the thrips-susceptible plant. RNA-sequencing analysis identified differentially expressed genes (DEGs) by comparing genome-wide gene expression changes between thrips-resistant and -susceptible eggplants. Consistent with metabolomic analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs revealed that the starch and sucrose metabolic pathway in which quinic acid is a metabolic by-product was highly enriched. External application of quinic acid enhances the resistance of susceptible eggplant to thrips. CONCLUSION: Our results showed that quinic acid plays a key role in the resistance to thrips. These findings highlight a potential application of quinic acid as a biocontrol agent to manage thrips and expand our knowledge to breed thrips-resistant eggplant. © 2022 Society of Chemical Industry.

Transcriptome Analysis of Fusarium-Tomato Interaction Based on an Updated Genome Annotation of Fusarium oxysporum f. sp. lycopersici Identifies Novel Effector Candidates That Suppress or Induce Cell Death in Nicotiana benthamiana.

Fusarium oxysporum f. sp. lycopersici (Fol) causes vascular wilt disease in tomato. Upon colonization of the host, Fol secretes many small effector proteins into the xylem sap to facilitate infection. Besides known SIX (secreted in xylem) proteins, the identity of additional effectors that contribute to Fol pathogenicity remains largely unexplored. We performed a deep RNA-sequencing analysis of Fol race 2-infected tomato, used the sequence data to annotate a published genome assembly generated via PacBio SMRT sequencing of the Fol race 2 reference strain Fol4287, and analysed the resulting transcriptome to identify Fol effector candidates among the newly annotated genes. We examined the Fol-infection expression profiles of all 13 SIX genes present in Fol race 2 and identified 27 new candidate effector genes that were likewise significantly upregulated upon Fol infection. Using Agrobacterium-mediated transformation, we tested the ability of 22 of the new candidate effector genes to suppress or induce cell death in leaves of Nicotiana benthamiana. One effector candidate designated Fol-EC19, encoding a secreted guanyl-specific ribonuclease, was found to trigger cell death and two effector candidates designated Fol-EC14 and Fol-EC20, encoding a glucanase and a secreted trypsin, respectively, were identified that can suppress Bax-mediated cell death. Remarkably, Fol-EC14 and Fol-EC20 were also found to suppress I-2/Avr2- and I/Avr1-mediated cell death. Using the yeast secretion trap screening system, we showed that these three biologically-active effector candidates each contain a functional signal peptide for protein secretion. Our findings provide a basis for further understanding the virulence functions of Fol effectors.

Optimized Production of Disulfide-Bonded Fungal Effectors in Escherichia coli Using CyDisCo and FunCyDisCo Coexpression Approaches.

Effectors are a key part of the arsenal of plant-pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognized by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterize effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a coexpression system within the heterologous host Escherichia coli, termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalized coexpression approach termed FunCyDisCo (Fungi CyDisCo) can significantly improve the production yields of numerous disulfide-bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallization of several of these reported fungal effectors. We suggest this approach could be broadly useful in the investigation of the function and recognition of a broad range of disulfide bond-containing effectors.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Nicotiana benthamiana asparagine synthetase associates with IP-L and confers resistance against tobacco mosaic virus via the asparagine-induced salicylic acid signalling pathway.

Asparagine synthetase is a key enzyme that catalyses the conversion of amide groups from glutamine or ammonium to aspartate, which leads to the generation of asparagine. However, the role of asparagine synthetase in plant immunity remains largely unknown. Here, we identified a Nicotiana benthamiana asparagine synthetase B (NbAS-B) that associates with tomato mosaic virus coat protein-interacting protein L (IP-L) using the yeast two-hybrid assay and examined its role in tobacco mosaic virus (TMV) resistance. The association of IP-L with NbAS-B was further confirmed by in vivo co-immunoprecipitation, luciferase complementation imaging, and bimolecular fluorescence complementation assays. IP-L and NbAS-B interact in the nucleus and cytosol and IP-L apparently stabilizes NbAS-B, thus enhancing its accumulation. The expressions of IP-L and NbAS-B are continuously induced on TMV-green fluorescent protein (GFP) infection. Co-silencing of IP-L and NbAS-B facilitates TMV-GFP infection. Overexpression of NbAS-B in tobacco reduces TMV-GFP infection by significantly improving the synthesis of asparagine. Furthermore, the external application of asparagine significantly inhibits the infection of TMV-GFP by activating the salicylic acid signalling pathway. These findings hold the potential for the future application of asparagine in the control of TMV.

Wheat Apoplast-Localized Lipid Transfer Protein TaLTP3 Enhances Defense Responses Against Puccinia triticina.

Plant apoplast serves as the frontier battlefield of plant defense in response to different types of pathogens. Many pathogenesis-related (PR) proteins are accumulated in apoplastic space during the onset of plant-pathogen interaction, where they act to suppress pathogen infection. In this study, we found the expression of Triticum aestivum lipid transfer protein 3 (TaLTP3) gene was unregulated during incompatible interaction mediated by leaf rust resistance genes Lr39/41 at the early infection stage. Stable transgenic wheat lines overexpressing TaLTP3 exhibited enhanced resistance to leaf rust pathogen Puccinia triticina. Transcriptome analysis revealed that overexpression of TaLTP3 specifically activated the transcription of pathogenesis-related protein 1a (TaPR1a) and multiple plant hormone pathways, including salicylic acid (SA), jasmonic acid (JA), and auxin, in response to the infection of the model bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Further investigation indicated that TaLTP3 physically associated with wheat TaPR1a protein in the apoplast. Transgenic wheat lines overexpressing TaLTP3 and TaPR1a showed higher accumulations of reactive oxygen species (ROS) during plant defense responses. All these findings suggested that TaLTP3 is involved in wheat resistance against leaf rust pathogen infection and forming a TaLTP3-TaPR1a complex in apoplast against this pathogen, which provides new insights into the functional roles of PR proteins.

Co-infection by Soil-Borne Fungal Pathogens Alters Disease Responses Among Diverse Alfalfa Varieties.

Fusarium oxysporum f. sp. medicaginis (Fom) and Rhizoctonia solani (Rs) are the major soil-borne fungal pathogens that pose severe threats to commercial alfalfa production in China. However, the effects of Fom and Rs co-infection on alfalfa and whether co-infection alters disease resistance responses among diverse varieties remain unknown. A collection of 80 alfalfa varieties (Medicago sativa) originated from seven countries were used to study the effects of Fom and Rs co-infection on alfalfa and host resistance responses. The co-infection resulted in more severe disease and reductions in growth and biomass allocation across varieties in comparison with either single infection by Fom or Rs; in addition, root morphology was much more strongly altered by the co-infection. Principal component analysis based on all plant traits showed that varieties under the co-infection were related to the single infection by Rs, being separated from Fom, and hierarchical clustering found differential response patterns among varieties upon co-infection compared with either single infection, with most varieties being highly susceptible to the co-infection. Furthermore, varieties that were most resistant to either single infection were not effective to co-infection, and there was no individual variety with resistance to both pathogens singly and co-infected. This study reveals for the first time that the co-infection by Fom and Rs alters disease resistance responses among diverse alfalfa varieties and provides useful information for developing alfalfa varieties with resistance to the co-occurrence of different soil-borne pathogens.

Synergistic Action of Commercially Available Fungicides for Protecting Wheat from Common Root Rot Caused by Bipolaris sorokiniana in China.

Wheat (Triticum aestivum) common root rot (CRR) caused by predominant fungal pathogen Bipolaris sorokiniana occurs in all wheat-growing regions worldwide and is difficult to control. In this study, the efficacy of eight fungicides against Bipolaris sorokiniana was examined in in vitro assays, and we determined that the combined application of two fungicides significantly inhibits the growth of fungal mycelium. Half of the maximal effective concentration of a mixture containing fludioxonil and difenoconazole in the ratio 1:4 was 0.0372 mg/liter, and the cotoxicity coefficient was 160.14. Under an environmentally controlled pot assay, seed treatment with the mixture of fludioxonil and difenoconazole in the 1:4 ratio demonstrated the best control efficiency at seedling and adult stages, respectively. The best synergistic mixture on seed treatment was assessed in a 2-year field experiment at Hebei, China. The best control efficacy achieved at the seedling and adult stages was 82.65% and 68.48%, respectively. Overall, the in vitro mycelial growth inhibition assay and controlled-environment and field studies indicated that the synergistic action of a mixture of fludioxonil and difenoconazole provides effective control against wheat CRR. These findings highlight the potential application of the fungicide combination for controlling CRR and reducing the selection pressure on fungal pathogens by lessening the use of various fungicides in the field.

Receptor-Like Kinases BAK1 and SOBIR1 Are Required for Necrotizing Activity of a Novel Group of Sclerotinia sclerotiorum Necrosis-Inducing Effectors.

Sclerotinia sclerotiorum is a characteristic necrotrophic plant pathogen and is dependent on the induction of host cell death for nutrient acquisition. To identify necrosis-inducing effectors, the genome of S. sclerotiorum was scanned for genes encoding small, secreted, cysteine-rich proteins. These potential effectors were tested for their ability to induce necrosis in Nicotiana benthamiana via Agrobacterium-mediated expression and for cellular localization in host cells. Six novel proteins were discovered, of which all but one required a signal peptide for export to the apoplast for necrotizing activity. Virus-induced gene silencing revealed that the five necrosis-inducing effectors with a requirement for secretion also required the plant co-receptor-like kinases Brassinosteroid Insensitive 1-Associated Receptor Kinase 1 (BAK1) and Suppressor of BAK1-Interacting Receptor-like Kinase 1 (SOBIR1) for the induction of necrosis. S. sclerotiorum necrosis-inducing effector 2 (SsNE2) represented a new class of necrosis-inducing proteins as orthologs were identified in several other phytopathogenic fungi that were also capable of inducing necrosis. Substitution of conserved cysteine residues with alanine reduced, but did not abolish, the necrotizing activity of SsNE2 and full-length protein was required for function as peptides spanning the entire protein were unable to induce necrosis. These results illustrate the importance of necrosis-inducing effectors for S. sclerotiorum virulence and the role of host extracellular receptor(s) in effector-triggered susceptibility to this pathogen.

The Brassica napus wall-associated kinase-like (WAKL) gene Rlm9 provides race-specific blackleg resistance.

In plants, race-specific defence against microbial pathogens is facilitated by resistance (R) genes which correspond to specific pathogen avirulence genes. This study reports the cloning of a blackleg R gene from Brassica napus (canola), Rlm9, which encodes a wall-associated kinase-like (WAKL) protein, a newly discovered class of race-specific plant RLK resistance genes. Rlm9 provides race-specific resistance against isolates of Leptosphaeria maculans carrying the corresponding avirulence gene AvrLm5-9, representing only the second WAKL-type R gene described to date. The Rlm9 protein is predicted to be cell membrane-bound and while not conclusive, our work did not indicate direct interaction with AvrLm5-9. Rlm9 forms part of a distinct evolutionary family of RLK proteins in B. napus, and while little is yet known about WAKL function, the Brassica-Leptosphaeria pathosystem may prove to be a model system by which the mechanism of fungal avirulence protein recognition by WAKL-type R genes can be determined.

Genome-wide analysis of NDR1/HIN1-like genes in pepper (Capsicum annuum L.) and functional characterization of CaNHL4 under biotic and abiotic stresses.

Plant NDR1/HIN1-like (NHL) genes play an important role in triggering plant defenses in response to biotic stresses. In this study, we performed a genome-wide identification of the NHL genes in pepper (Capsicum annuum L.) and characterized the functional roles of these CaNHL genes in response to abiotic stresses and infection by different pathogens. Phylogenetic analysis revealed that CaNHLs can be classified into five distinct subgroups, with each group containing generic and specific motifs. Regulatory element analysis showed that the majority of the promoter regions of the identified CaNHLs contain jasmonic acid (JA)-responsive and salicylic acid (SA)-responsive elements, and transcriptomic analysis revealed that CaNHL genes are expressed in all the examined tissues of pepper. The CaNHL1, CaNHL4, CaNHL6, CaNHL10, CaNHL11, and CaNHL12 genes were significantly upregulated under abiotic stress as well as in response to different pathogens, such as TMV, Phytophthora capsici and Pseudomonas syringae. In addition, we found that CaNHL4 localizes to the plasma membrane. CaNHL4-silenced pepper plants display significantly increased susceptibility to TMV, Phytophthora capsici and Pseudomonas syringae, exhibiting reduced expression of JA-related and SA-related genes and reduced ROS production. However, transient overexpression of CaNHL4 in pepper increases the expression of JA-related and SA-related genes, enhances the accumulation of ROS, and inhibits the infection of these three pathogens. Collectively, for the first time, we identified the NHL genes in pepper and demonstrated that CaNHL4 is involved in the production of ROS and that it also regulates the expression of JA-related and SA-related genes in response to different pathogens, suggesting that members of the CaNHL family play an essential role in the disease resistance of pepper.

Synthetic chloroinconazide compound exhibits highly efficient antiviral activity against tobacco mosaic virus.

BACKGROUND: Development of anti-plant-virus compounds and improvement of biosafety remain hot research topics in controlling plant viral disease. Tobacco mosaic virus (TMV) infects all tobacco species as well as many other plants worldwide and causes severe losses in tobacco production. To date, no efficient chemical treatments are known to protect plants from virus infection. Therefore, the search for a highly active antiviral compound with high efficacy in field application is required. RESULTS: We reported the synthesis of a novel antiviral halogenated acyl compound Chloroinconazide (CHI) using tryptophan as a substrate and examined its anti-TMV activity. We found that CHI displayed the ability to strongly inhibit the infection of TMV on Nicotiana benthamiana via multiple mechanisms. We observed that CHI was able to impair the virulence of TMV by directly altering the morphological structure of virions and increasing the activity of anti-oxidative enzymes, resulting in reduced TMV-induced ROS production during infection of the plant. In addition, the expression of salicylic acid-responsive genes was significantly increased after CHI application. However, after application of CHI on SA-deficient NahG plants no obvious anti-TMV activity was observed, suggesting that the SA signaling pathway was required for CHI-induced anti-TMV activity associated with reduced infection of TMV. CHI exhibited no effects on plant growth and development. CONCLUSION: The easily synthesized CHI can actively induce plant resistance against TMV as well as act on virus particles and exhibits high biosafety, which provides a potential for commercial application of CHI in controlling plant virus disease in the future. © 2020 Society of Chemical Industry.