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.

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.

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.

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.

Mimicking the Host Regulation of Salicylic Acid: A Virulence Strategy by the Clubroot Pathogen Plasmodiophora brassicae.

The plant hormone salicylic acid (SA) plays a critical role in defense against biotrophic pathogens such as Plasmodiophora brassicae, which is an obligate pathogen of crucifer species and the causal agent of clubroot disease of canola (Brassica napus). P. brassicae encodes a protein, predicted to be secreted, with very limited homology to benzoic acid (BA)/SA-methyltransferase, designated PbBSMT. PbBSMT has a SA- and an indole-3-acetic acid-binding domain, which are also present in Arabidopsis thaliana BSMT1 (AtBSMT1) and, like AtBSMT1, has been shown to methylate BA and SA. In support of the hypothesis that P. brassicae uses PbBSMT to overcome SA-mediated defenses by converting SA into inactive methyl salicylate (MeSA), here, we show that PbBSMT suppresses local defense and provide evidence that PbBSMT is much more effective than AtBSMT1 at suppressing the levels of SA and its associated effects. Basal SA levels in Arabidopsis plants that constitutively overexpress PbBSMT compared with those in Arabidopsis wild-type Col-0 (WT) were reduced approximately 80% versus only a 50% reduction in plants overexpressing AtBSMT1. PbBSMT-overexpressing plants were more susceptible to P. brassicae than WT plants; they also were partially compromised in nonhost resistance to Albugo candida. In contrast, AtBSMT1-overexpressing plants were not more susceptible than WT to either P. brassicae or A. candida. Furthermore, transgenic Arabidopsis and tobacco plants overexpressing PbBSMT exhibited increased susceptibility to virulent Pseudomonas syringae pv. tomato DC3000 (DC3000) and virulent Pseudomonas syringae pv. tabaci, respectively. Gene-mediated resistance to DC3000/AvrRpt2 and tobacco mosaic virus (TMV) was also compromised in Arabidopsis and Nicotiana tabacum 'Xanthi-nc' plants overexpressing PbBSMT, respectively. Transient expression of PbBSMT or AtBSMT1 in lower leaves of N. tabacum Xanthi-nc resulted in systemic acquired resistance (SAR)-like enhanced resistance to TMV in the distal systemic leaves. Chimeric grafting experiments revealed that, similar to SAR, the development of a PbBSMT-mediated SAR-like phenotype was also dependent on the MeSA esterase activity of NtSABP2 in the systemic leaves. Collectively, these results strongly suggest that PbBSMT is a novel effector, which is secreted by P. brassicae into its host plant to deplete pathogen-induced SA accumulation.

Functional analysis of a pathogenesis-related thaumatin-like protein gene TaLr35PR5 from wheat induced by leaf rust fungus.

BACKGROUND: Plants have evolved multifaceted defence mechanisms to resist pathogen infection. Production of the pathogenesis-related (PR) proteins in response to pathogen attack has been implicated in plant disease resistance specialized in systemic-acquired resistance (SAR). Our earlier studies have reported that a full length TaLr35PR5 gene, encoding a protein exhibiting amino acid and structural similarity to a sweet protein thaumatin, was isolated from wheat near-isogenic line TcLr35. The present study aims to understand the function of TaLr35PR5 gene in Lr35-mediated adult resistance to Puccinia triticina. RESULTS: We determined that the TaLr35PR5 protein contained a functional secretion peptide by utilizing the yeast signal sequence trap system. Using a heterologous expression assay on onion epidermal cells we found that TaLr35PR5 protein was secreted into the apoplast of onion cell. Expression of TaLr35PR5 was significantly reduced in BSMV-induced gene silenced wheat plants, and pathology test on these silenced plants revealed that Lr35-mediated resistance phenotype was obviously altered, indicating that Lr35-mediated resistance was compromised. CONCLUSIONS: All these findings strongly suggest that TaLr35PR5 is involved in Lr35-mediated adult wheat defense in response to leaf rust attack.

Genome sequence of the potato pathogenic fungus Alternaria solani HWC-168 reveals clues for its conidiation and virulence.

BACKGROUND: Alternaria solani is a known air-born deuteromycete fungus with a polycyclic life cycle and is the causal agent of early blight that causes significant yield losses of potato worldwide. However, the molecular mechanisms underlying the conidiation and pathogenicity remain largely unknown. RESULTS: We produced a high-quality genome assembly of A. solani HWC-168 that was isolated from a major potato-producing region of Northern China, which facilitated a comprehensive gene annotation, the accurate prediction of genes encoding secreted proteins and identification of conidiation-related genes. The assembled genome of A. solani HWC-168 has a genome size 32.8 Mb and encodes 10,358 predicted genes that are highly similar with related Alternaria species including Alternaria arborescens and Alternaria brassicicola. We identified conidiation-related genes in the genome of A. solani HWC-168 by searching for sporulation-related homologues identified from Aspergillus nidulans. A total of 975 secreted protein-encoding genes, which might act as virulence factors, were identified in the genome of A. solani HWC-168. The predicted secretome of A. solani HWC-168 possesses 261 carbohydrate-active enzymes (CAZy), 119 proteins containing RxLx[EDQ] motif and 27 secreted proteins unique to A. solani. CONCLUSIONS: Our findings will facilitate the identification of conidiation- and virulence-related genes in the genome of A. solani. This will permit new insights into understanding the molecular mechanisms underlying the A. solani-potato pathosystem and will add value to the global fungal genome database.

Development of a Semi-nested PCR-Based Method for Specific and Rapid Detection of Alternaria solani Causing Potato Early Blight in Soil.

Early blight, caused by Alternaria solani, is one of the most devastating diseases of potato that causes severe yield loss worldwide. The infected potato debris existed in the soil serve as the initial infection sources for the next growing potato. Current identification of A. solani in soil relies primarily on cultural and morphological characteristics, which are time-consuming and inaccurate. In this study, a semi-nested PCR method was developed using primers based on internal transcribed spacer region that is specific to A. solani. 20 isolates including 6 Alternaria species and 10 other species of common potato pathogens were used to examine the specificity of the primers. The primer set ptAsQ-F/ptAs-R was highly specific to A. solani, as a product of 251 bp was amplified only from A. solani isolates and no amplification signal was observed from other tested species. The sensitivity of this method determined using A. solani genomic DNA was 10 fg. This PCR assay was also successfully employed to detect A. solani in soil with the detection sensitivity of one conidia spore in 0.5 g of soil. To the best of our knowledge, this is the first report of molecular detection of A. solani in soil, which provides a useful tool for early and rapid detection of early blight in soil before next growing season.

The Brassica napus receptor-like protein RLM2 is encoded by a second allele of the LepR3/Rlm2 blackleg resistance locus.

Leucine-rich repeat receptor-like proteins (LRR-RLPs) are highly adaptable parts of the signalling apparatus for extracellular detection of plant pathogens. Resistance to blackleg disease of Brassica spp. caused by Leptosphaeria maculans is largely governed by host race-specific R-genes, including the LRR-RLP gene LepR3. The blackleg resistance gene Rlm2 was previously mapped to the same genetic interval as LepR3. In this study, the LepR3 locus of the Rlm2 Brassica napus line 'Glacier DH24287' was cloned, and B. napus transformants were analysed for recovery of the Rlm2 phenotype. Multiple B. napus, B. rapa and B. juncea lines were assessed for sequence variation at the locus. Rlm2 was found to be an allelic variant of the LepR3 LRR-RLP locus, conveying race-specific resistance to L. maculans isolates harbouring AvrLm2. Several defence-related LRR-RLPs have previously been shown to associate with the RLK SOBIR1 to facilitate defence signalling. Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation of RLM2-SOBIR1 studies revealed that RLM2 interacts with SOBIR1 of Arabidopsis thaliana when co-expressed in Nicotiana benthamiana. The interaction of RLM2 with AtSOBIR1 is suggestive of a conserved defence signalling pathway between B. napus and its close relative A. thaliana.

The AVR2-SIX5 gene pair is required to activate I-2-mediated immunity in tomato.

Plant-invading microbes betray their presence to a plant by exposure of antigenic molecules such as small, secreted proteins called 'effectors'. In Fusarium oxysporum f. sp. lycopersici (Fol) we identified a pair of effector gene candidates, AVR2-SIX5, whose expression is controlled by a shared promoter. The pathogenicity of AVR2 and SIX5 Fol knockouts was assessed on susceptible and resistant tomato (Solanum lycopersicum) plants carrying I-2. The I-2 NB-LRR protein confers resistance to Fol races carrying AVR2. Like Avr2, Six5 was found to be required for full virulence on susceptible plants. Unexpectedly, each knockout could breach I-2-mediated disease resistance. So whereas Avr2 is sufficient to induce I-2-mediated cell death, Avr2 and Six5 are both required for resistance. Avr2 and Six5 interact in yeast two-hybrid assays as well as in planta. Six5 and Avr2 accumulate in xylem sap of plants infected with the reciprocal knockouts, showing that lack of I-2 activation is not due to a lack of Avr2 accumulation in the SIX5 mutant. The effector repertoire of a pathogen determines its host specificity and its ability to manipulate plant immunity. Our findings challenge an oversimplified interpretation of the gene-for-gene model by showing requirement of two fungal genes for immunity conferred by one resistance gene.

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.

MITEs in the promoters of effector genes allow prediction of novel virulence genes in Fusarium oxysporum.

BACKGROUND: The plant-pathogenic fungus Fusarium oxysporum f.sp.lycopersici (Fol) has accessory, lineage-specific (LS) chromosomes that can be transferred horizontally between strains. A single LS chromosome in the Fol4287 reference strain harbors all known Fol effector genes. Transfer of this pathogenicity chromosome confers virulence to a previously non-pathogenic recipient strain. We hypothesize that expression and evolution of effector genes is influenced by their genomic context. RESULTS: To gain a better understanding of the genomic context of the effector genes, we manually curated the annotated genes on the pathogenicity chromosome and identified and classified transposable elements. Both retro- and DNA transposons are present with no particular overrepresented class. Retrotransposons appear evenly distributed over the chromosome, while DNA transposons tend to concentrate in large chromosomal subregions. In general, genes on the pathogenicity chromosome are dispersed within the repeat landscape. Effector genes are present within subregions enriched for DNA transposons. A miniature Impala (mimp) is always present in their promoters. Although promoter deletion studies of two effector gene loci did not reveal a direct function of the mimp for gene expression, we were able to use proximity to a mimp as a criterion to identify new effector gene candidates. Through xylem sap proteomics we confirmed that several of these candidates encode proteins secreted during plant infection. CONCLUSIONS: Effector genes in Fol reside in characteristic subregions on a pathogenicity chromosome. Their genomic context allowed us to develop a method for the successful identification of novel effector genes. Since our approach is not based on effector gene similarity, but on unique genomic features, it can easily be extended to identify effector genes in Fo strains with different host specificities.

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.

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.

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.

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.

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.

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.