Potential Involvement of MnCYP710A11 in Botrytis cinerea Resistance in Arabidopsis thaliana and Morus notabilis.

Cytochrome P450 (CYP) is a crucial oxidoreductase enzyme that plays a significant role in plant defense mechanisms. In this study, a specific cytochrome P450 gene (MnCYP710A11) was discovered in mulberry (Morus notabilis). Bioinformatic analysis and expression pattern analysis were conducted to elucidate the involvement of MnCYP710A11 in combating Botrytis cinerea infection. After the infection of B. cinerea, there was a notable increase in the expression of MnCYP710A11. MnCYP710A11 is overexpressed in Arabidopsis and mulberry and strongly reacts to B. cinerea. The overexpression of the MnCYP710A11 gene in Arabidopsis and mulberry led to a substantial enhancement in resistance against B. cinerea, elevated catalase (CAT) activity, increased proline content, and reduced malondialdehyde (MDA) levels. At the same time, H2O2 and O2- levels in MnCYP710A11 transgenic Arabidopsis were decreased, which reduced the damage of ROS accumulation to plants. Furthermore, our research indicates the potential involvement of MnCYP710A11 in B. cinerea resistance through the modulation of other resistance-related genes. These findings establish a crucial foundation for gaining deeper insights into the role of cytochrome P450 in mulberry plants.

Transcriptomic analysis and knockout experiments reveal the role of suhB in the biocontrol effects of Pantoea jilinensis D25 on Botrytis cinerea.

Tomato gray mold, caused by Botrytis cinerea, is an important disease in tomato. Pantoea jilinensis D25, isolated form tomato rhizosphere soil, can prevent B. cinerea infection in tomato. To determine the underlying biocontrol mechanism, the transcriptome of P. jilinensis D25 was assessed. Differential expression analysis revealed that 941 genes were upregulated and 997 genes were downregulated. Through transcriptome analysis, the suhB gene was knocked out. ΔPj-suhB exhibited lower swimming motility and colonization abilities than strain D25. After 4 days of co-cultivation, ΔPj-suhB could reduce the colony diameter, mycelial weight, and spore production of B. cinerea with the inhibitory rates of 31.72 %, 39.62 %, and 47.42 %, respectively, compared with control. However, the inhibitory rates of strain D25 were 52.91 %, 60.09 %, and 76.85 %, respectively, compared with control. Strain D25 could significantly downregulate pathogenesis-related genes in B. cinerea, whereas the expression level of these genes in B. cinerea was higher after treatment with ΔPj-suhB than after that with strain D25. In vitro experiments revealed that the lesion area and disease control efficacy were 1.520 and 0.038 cm2 and 68.7 % and 99.0 %, respectively, after ΔPj-suhB and strain D25 treatments. Pot experiments revealed that ΔPj-suhB and strain D25 could prevent tomato plants from B. cinerea infection with the disease reduction rate of 37.5 % and 75.0 %, respectively. Though the activities of defense-related enzymes and expression level of defense related genes in tomato plants were increased under ΔPj-suhB treatment, these effects were higher after strain D25 treatment. Thus, these results demonstrated that suhB was the key gene in strain D25 underlying its biocontrol effect and mobility.

AtRAC7/ROP9 Small GTPase Regulates A. thaliana Immune Systems in Response to B. cinerea Infection.

Botrytis cinerea is a necrotrophic fungus that can cause gray mold in over 1400 plant species. Once it is detected by Arabidopsis thaliana, several defense responses are activated against this fungus. The proper activation of these defenses determines plant susceptibility or resistance. It has been proposed that the RAC/ROP small GTPases might serve as a molecular link in this process. In this study, we investigate the potential role of the Arabidopsis RAC7 gene during infection with B. cinerea. For that, we evaluated A. thaliana RAC7-OX lines, characterized by the overexpression of the RAC7 gene. Our results reveal that these RAC7-OX lines displayed increased susceptibility to B. cinerea infection, with enhanced fungal colonization and earlier lesion development. Additionally, they exhibited heightened sensitivity to bacterial infections caused by Pseudomonas syringae and Pectobacterium brasiliense. By characterizing plant canonical defense mechanisms and performing transcriptomic profiling, we determined that RAC7-OX lines impaired the plant transcriptomic response before and during B. cinerea infection. Global pathway analysis of differentially expressed genes suggested that RAC7 influences pathogen perception, cell wall homeostasis, signal transduction, and biosynthesis and response to hormones and antimicrobial compounds through actin filament modulation. Herein, we pointed out, for first time, the negative role of RAC7 small GTPase during A. thaliana-B. cinerea interaction.

Antifungal activity and mechanism of palmarosa essential oil against pathogen Botrytis cinerea in the postharvest onions.

AIMS: Botrytis cinerea is a pathogenic fungus that infests multiple crops, which causes a severe decrease in yield and generates substantial losses in the economy. Palmarosa essential oil (PEO) is a primary aromatic compound extracted from palmarosa that is commonly used for scent, medicine, and flavoring foods due to its diverse bioactive properties. In this study, we explored the antifungal activity and the main mechanism of action of PEO against B. cinerea. In addition, the components and control effects of PEO were also studied. METHODS AND RESULTS: The antifungal assay was tested using the mycelial growth rate method and colony morphology. The constituents of PEO were identified according to gas chromatography/mass spectrometry (GC-MS). The main mechanism of action of PEO was evaluated by measuring representative indicators, which consist of cell contents leakage, excess reactive oxygen species (ROS), and other related indicators. The results indicated that at a concentration of 0.60 ml l-1, PEO exhibits strong antifungal activity against B. cinerea. The PEO mainly included 13 compounds, of which citronellol (44.67%), benzyl benzoate (14.66%), and acetyl cedrene (9.63%) might be the main antifungal ingredients. The study elucidated the main mechanism of action of PEO against B. cinerea, which involved the disruption of cell membrane structure, resulting in altered the cell membrane permeability, leakage of cell contents, and accumulation of excess ROS. CONCLUSIONS: PEO is a satisfactory biological control agent that inhibits B. cinerea in postharvest onions. PEO (0.60 ml l-1) exhibited strong antifungal activity by disrupting the cell membrane structure, altering cell membrane permeability, leading to the cell contents leakage, accumulation of excess ROS and increased level of Malondialdehyde (MDA) compared to the control group.

Mulberry MnGolS2 Mediates Resistance to Botrytis cinerea on Transgenic Plants.

Galactitol synthetase (GolS) as a key enzyme in the raffinose family oligosaccharides (RFOs) biosynthesis pathway, which is closely related to stress. At present, there are few studies on GolS in biological stress. The expression of MnGolS2 gene in mulberry was increased under Botrytis cinerea infection. The MnGolS2 gene was cloned and ectopically expressed in Arabidopsis. The content of MDA in leaves of transgenic plants was decreased and the content of CAT was increased after inoculation with B. cinerea. In this study, the role of MnGolS2 in biotic stress was demonstrated for the first time. In addition, it was found that MnGolS2 may increase the resistance of B. cinerea by interacting with other resistance genes. This study offers a crucial foundation for further research into the role of the GolS2 gene.

Unraveling the Molecular Mechanisms of Tomatoes' Defense against Botrytis cinerea: Insights from Transcriptome Analysis of Micro-Tom and Regular Tomato Varieties.

Botrytis cinerea is a devastating fungal pathogen that causes severe economic losses in global tomato cultivation. Understanding the molecular mechanisms driving tomatoes' response to this pathogen is crucial for developing effective strategies to counter it. Although the Micro-Tom (MT) cultivar has been used as a model, its stage-specific response to B. cinerea remains poorly understood. In this study, we examined the response of the MT and Ailsa Craig (AC) cultivars to B. cinerea at different time points (12-48 h post-infection (hpi)). Our results indicated that MT exhibited a stronger resistant phenotype at 18-24 hpi but became more susceptible to B. cinerea later (26-48 hpi) compared to AC. Transcriptome analysis revealed differential gene expression between MT at 24 hpi and AC at 22 hpi, with MT showing a greater number of differentially expressed genes (DEGs). Pathway and functional annotation analysis revealed significant differential gene expression in processes related to metabolism, biological regulation, detoxification, photosynthesis, and carbon metabolism, as well as some immune system-related genes. MT demonstrated an increased reliance on Ca2+ pathway-related proteins, such as CNGCs, CDPKs, and CaMCMLs, to resist B. cinerea invasion. B. cinerea infection induced the activation of PTI, ETI, and SA signaling pathways, involving the modulation of various genes such as FLS2, BAK1, CERK1, RPM, SGT1, and EDS1. Furthermore, transcription factors such as WRKY, MYB, NAC, and AUX/IAA families played crucial regulatory roles in tomatoes' defense against B. cinerea. These findings provide valuable insights into the molecular mechanisms underlying tomatoes' defense against B. cinerea and offer potential strategies to enhance plant resistance.

Genome-wide identification and validation of tomato-encoded sRNA as the cross-species antifungal factors targeting the virulence genes of Botrytis cinerea.

Recent evidence shows that small RNAs are transferred from a species to another through cross-species transmission and exhibit biological activities in the receptor. In this study, we focused on tomato-derived sRNAs play a role of defense against Botrytis cinerea. Bioinformatics method was firstly employed to identify tomato-encoded sRNAs as the cross-species antifungal factors targeting B. cinerea genes. Then the expression levels of some identifed sRNAs were checked in B. cinerea-infected plant using qRT-PCR method. Exogenic RNA-induced gene silences analysis were performed to investigate the antifungal roles of the sRNAs, and the target genes in B. cinerea of antifungal sRNAs would be confirmed by using co-expression analysis. Results showed that a total of 21 B.cinerea-induced sRNAs with high abundance were identified as the cross-kingdom regulator candidates. Among them, three sRNAs containing a miRNA (miR396a-5p) and two siRNA (siR3 and siR14) were selected for experimental validation and bioassay analysis. qRT-PCR confirmed that all of these 3 sRNAs were induced in tomato leaves by B. cinerea infection. Correspondingly, 4 virulence genes of B. cinerea respectively targeted by these 3 sRNAs were down-regulated. Bioassay revealed that all of these 3 cross-species sRNAs could inhibit the virulence and spore gemination of B. cinerea. Correspondingly, the coding genes of B. cinerea targeted by these sRNAs were also down-regulated. Moreover, the virulence inhibition by double strand sRNA was more effective than that by single strand sRNA. The inhibition efficiency of sRNA against B. cinerea increased with the increase of its concentration. Our findings provide new evidence into the coevolution of pathogens and host plants, as well as new directions for the use of plant-derived sRNAs to control pathogens.

Effects of Intermittent Temperature and Humidity Regulation on Tomato Gray Mold.

Temperature and humidity play an important role in plant-pathogen interactions. However, regulating the temperature and humidity specifically to inhibit the development of plant diseases remains unclear. In this study, we explored the influence of intermittent temperature and humidity variation on tomato gray mold. Intermittent regulation of temperature and humidity (increasing temperature with decreasing humidity for different periods within 24 h) inhibited the disease severity of plants and the infection process of Botrytis cinerea. The 4-h treatment (increasing temperature accompanied by decreasing humidity for 4 h and recovering for 4 h, and so on) effectively inhibited the development of tomato gray mold, reduced the biomass of B. cinerea, delayed the differentiation time of mycelia, and inhibited the accumulation of hydrogen peroxide in tomato leaves at the later stage of infection. The increased expressions of heat-shock protein (HSP) genes HSP20, HSP70, HSP90, BAG6, and BAG7 in tomato were mainly caused by environmental changes and environment-plant-pathogen interactions, and the increased expression of the latter was greater than that of the former in the 2-h (increasing temperature accompanied by decreasing humidity for 2 h and recovering for 2 h, and so on) and 4-h treatments. Pathogen infection induced the expression of defense-related genes in tomato, and the increase in the expressions of FLS2, FEI1, PI2, Pti5, and WRKY75 induced by B. cinerea in the 4-h treatment was greater than that under unregulated temperature and humidity conditions. In general, intermittent temperature and humidity variation can effectively inhibit the development of tomato gray mold, and the 4-h treatment had the best inhibitory effect.

MnASI1 Mediates Resistance to Botrytis cinerea in Mulberry (Morus notabilis).

Six α-amylase/subtilisin inhibitor genes (MnASIs) were identified from mulberry (Morus notabilis). In this study, bioinformatics and expression pattern analysis of six MnASIs were performed to determine their roles in resistance to B. cinerea. The expression of all six MnASIs was significantly increased under Botrytis cinerea infection. MnASI1, which responded strongly to B. cinerea, was overexpressed in Arabidopsis and mulberry. The resistance of Arabidopsis and mulberry overexpressing MnASI1 gene to B. cinerea was significantly improved, the catalase (CAT) activity was increased, and the malondialdehyde (MDA) content was decreased after inoculation with B. cinerea. At the same time, H2O2 and O2- levels were reduced in MnASI1 transgenic Arabidopsis, reducing the damage of ROS accumulation to plants. In addition, MnASI1 transgenic Arabidopsis increased the expression of the salicylic acid (SA) pathway-related gene AtPR1. This study provides an important reference for further revealing the function of α-amylase/subtilisin inhibitors.

GABA keeps nitric oxide in balance by regulating GSNOR to enhance disease resistance of harvested tomato against Botrytis cinerea.

Gamma-aminobutyric acid (GABA), a widely distributed metabolite in prokaryotes and eukaryotes, has many functions for plants in stress responses. In this study, hypotonic treatment with 10 mmol L-1 GABA in cherry tomato induced resistance to Botrytis cinerea with markedly lower disease incidence and lesion diameter, led to endogenous nitric oxide (NO) tansient accumulation before inoculation the pathogen then decrease after inoculation, and enhanced the content of arginine (Arg) and glutamic acid (Glu). The resistance of fruit treated with a NO scavenger, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), was significantly reduced. Moreover, the enzyme activity and gene expression of S-nitrosoglutathione reductase (GSNOR) were enhanced following endogenous NO increased. The endogenous NO level was excessively high after treatment with a GSNOR scavenger, N6022, making the fruit more susceptible to pathogen. Similarly, after break down of SlGSNOR, fruit had much higher endogenous NO and lower disease resistance. However, overexpression of SlGSNOR exhibited opposite consequences. These results suggest that a suitable level of NO is beneficial for enhancing disease resistance, and GABA can help tomatoes maintain NO equilibrium by regulating GSNOR.

The Linker Region Promotes Activity and Binding Efficiency of Modular LPMO towards Polymeric Substrate.

Lytic polysaccharide monooxygenases (LPMOs) mediate oxidative degradation of plant polysaccharides. The genes encoding LPMOs are most commonly arranged with one catalytic domain, while a few are found tethered to additional noncatalytic units, i.e., cellulase linker and carbohydrate-binding module (CBM). The presence of CBM is known to facilitate catalysis by directing the enzymes toward cellulosic polymer, while the role of linkers is poorly understood. Based on limited experimental evidence, linkers are believed to serve merely as flexible spacers between the structured domains. Thus, this study aims to unravel the role of the linker regions present in LPMO sequences. For this, we analyzed the genome of Botrytis cinerea and found 9 genes encoding cellulose lytic monooxygenases (AA9 family), of which BcAA9C was overexpressed in cellulose-inducible conditions. We designed variants of flLPMO (full-length enzyme) with truncation of either linker or CBM to examine the role of linker in activity, binding, and thermal stability of the associated monooxygenase. Biochemical assays predicted that the deletion of linker does not impact the potential of flLPMO for catalyzing the oxidation of Amplex Red, but that it does have a major influence on the capability of flLPMO to degrade recalcitrant polysaccharide substrate. Langmuir isotherm and SEM analysis demonstrated that linker domain aids in polysaccharide binding during flLPMO-mediated deconstruction of plant cell wall. Interestingly, linker domain was also found to contribute toward the thermostability of flLPMO. Overall, our study reveals that linker is not merely a spacer, but plays a key role in LPMO-mediated biomass fibrillation; these findings are broadly applicable to other polysaccharide-degrading enzymes. IMPORTANCE The polysaccharide-disintegrating carbohydrate-active enzymes (CAZymes) are often found with multimodular architecture, where the catalytic domain is connected to an accessory CBM domain with the help of a flexible linker region. So far, the linker has been understood merely as a flexible spacer between the two domains. Therefore, the current study is designed to determine the role of linker in polysaccharide fibrillation. To conceive this study, we have selected LPMO as a model enzyme, as it is not only an industrially relevant enzyme but it also harbors a catalytic domain, linker region, and CBM domain. The present study highlighted the crucial and indispensable role of the linker region in mediating polysaccharide disintegration. Considering its role in binding, thermostability, and activity toward polysaccharide substrate, we propose linker as a potential candidate for future CAZyme engineering.

Arabidopsis thaliana Cuticle Composition Contributes to Differential Defense Response to Botrytis cinerea.

The chemical composition of a plant cuticle can change in response to various abiotic or biotic stresses and plays essential functions in disease resistance responses. Arabidopsis thaliana mutants altered in cutin content are resistant to Botrytis cinerea, presumably because of increased cuticular water and solute permeability, allowing for faster induction of defense responses. Within this context, our knowledge of wax mutants is limited against this pathogen. We tested the contribution of cuticular components to immunity to B. cinerea using mutants altered in either cutin or wax alone, or in both cutin and wax contents. We found that even all the tested mutants showed increased permeability and reactive oxygen species (ROS) accumulation in comparison with wild-type plants and that only cutin mutants showed resistance. To elucidate the early molecular mechanisms underlying cuticle-related immunity, we performed a transcriptomic analysis. A set of upregulated genes involved in cell wall integrity and accumulation of ROS were shared by the cutin mutants bdg, lacs2-3, and eca2, but not by the wax mutants cer1-4 and cer3-6. Interestingly, these genes have recently been shown to be required in B. cinerea resistance. In contrast, we found the induction of genes involved in abiotic stress shared by the two wax mutants. Our study reveals new insight that the faster recognition of a pathogen by changes in cuticular permeability is not enough to induce resistance to B. cinerea, as has previously been hypothesized. In addition, our data suggest that mutants with resistant phenotype can activate other defense pathways, different from those canonical immune ones.

Appraisal of Chitosan-Gum Arabic-Coated Bipolymeric Nanocarriers for Efficient Dye Removal and Eradication of the Plant Pathogen Botrytis cinerea.

The treatment of textile wastewater comprising many dyes as contaminants endures an essential task for environmental remediation. In addition, combating antifungal multidrug resistance (MDR) is an intimidating task, specifically owing to the limited options of alternative drugs with multitarget drug mechanisms. Incorporating natural polymeric biomaterials for drug delivery provides desirable properties for drug molecules, effectively eradicating MDR fungal growth. The current study fabricated the bipolymeric drug delivery system using chitosan-gum arabic-coated liposome 5ID nanoparticles (CS-GA-5ID-LP-NPs). This study focused on improving the solubility and sustained release profile of 5I-1H-indole (5ID). These NPs were characterized and tested mechanically as a dye adsorbent as well as their antifungal potencies against the plant pathogen, Botrytis cinerea. CS-GA-5ID-LP-NPs showed 71.23% congo red dye removal compared to crystal violet and phenol red from water and effectively had an antifungal effect on B. cinerea at 25 µg/mL MIC concentrations. The mechanism of the inhibition of B. cinerea via CS-GA-5ID-LP-NPs was attributed to stabilized microtubule polymerization in silico and in vitro. This study opens a new avenue for designing polymeric NPs as adsorbents and antifungal agents for environmental and agriculture remediation.

Transcriptional differentiation driving Cucumis sativus-Botrytis cinerea interactions based on the Skellam model and Bayesian networks.

Robust statistical tools such as the Skellam model and Bayesian networks can capture the count properties of transcriptome sequencing data and clusters of genes among treatments, thereby improving our knowledge of gene functions and networks. In this study, we successfully implemented a model to analyze a transcriptome dataset of Cucumis sativus and Botrytis cinerea before and after their interaction. First, 4200 differentially expressed genes (DEGs) from C. sativus were clustered into 17 distinct groups, and 670 DEGs from B. cinerea were clustered into 12 groups. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied on these DEGs to assess the interactions between C. sativus and B. cinerea. In C. sativus, more DEGs were divided into terms in the molecular function and biological process domains than into cellular components, and 277 DEGs were allocated to 19 KEGG pathways. In B. cinerea, more DEGs were divided into terms in the biological process and cellular component domains than into molecular functions, and 150 DEGs were allocated to 26 KEGG pathways. In this study, we constructed networks of genes that interact with each other to screen hub genes based on a directed graphical model known as Bayesian networks. Through a detailed GO analysis, we excavated hub genes which were biologically meaningful. These results verify that availability of Skellam model and Bayesian networks in clustering gene expression data and sorting out hub genes. These models are instrumental in increasing our knowledge of gene functions and networks in plant-pathogen interaction.

Study of UV-C treatments on postharvest life of blueberries 'O'Neal' and correlation between structure and quality parameters.

The effect of different doses of UV-C light (5.3, 8.3 and 11.4 kJ/m2) on native mycobiota and Botrytis cinerea incidence, micro and ultrastructure, biomechanical properties and weight loss of blueberry fruit cv. O'Neal during 20 days of storage at 8 ± 1 °C was evaluated. Decay incidence was significantly reduced by all UV-C light doses for both, native mycobiota and inoculated B. cinerea. The highest UV-C dose studied (11.4 kJ/m2) was the most effective indelaying the onset of fungal and B. cinerea infection (6 and 4 days, respectively). UV-C irradiation caused some distinctive changes in fruit structure characterized by redistribution, alteration and partial removal of epicuticular waxes, reinforcement of epicarp cell walls, and modifications in the cuticle. Biomechanical parameters were not affected by UV-C treatments excepting at day 15 where irradiated samples showed higher values of rupture force (FR) and deformation (D). Structure changes partially explained the significant increase in weight loss, FR and D values in irradiated fruit after 15 days of storage. UV-C irradiation could be an alternative for delaying and reducing fungal infection. However, postharvest shelf-life of irradiated blueberries could be limited by the negative effect on weight loss.

Over-expression of SlWRKY46 in tomato plants increases susceptibility to Botrytis cinerea by modulating ROS homeostasis and SA and JA signaling pathways.

WRKY, as one of the largest families of transcription factors (TFs), binds to cis-acting elements of downstream genes to regulate biotic and abiotic stress. However, the role of SlWRKY46 in fungal disease response induced by Botrytis cinerea (B.cinerea) and potential mechanism remains obscure. To ascertain the role of SlWRKY46 in response to B.cinerea, we constructed SlWRKY46-overexpression plants, which were then inoculated with B.cinerea. SlWRKY46-overexpression plants were more susceptible to B.cinerea and accompanied by the inhibited activities of phenylalanine ammonialyase (PAL), polyphenol oxidase (PPO), chitinase (CHI), and ß-1,3-glucanase (GLU). Additionally, SlWRKY46-overexpression plants showed the decreased activities of ascorbate peroxidase (APX), superoxide dismutase (SOD) and the content of H2O2, and the increased content of O2•-. Moreover, over-expression of SlWRKY46 suppressed the salicylic acid (SA) and jasmonic acid (JA) marker genes, pathogenesis related protein (PR1), and proteinase inhibitors (PI Ⅰ and PI Ⅱ) and consequently aggravated the disease symptoms. Therefore, we speculated that SlWRKY46 played negative regulatory roles in B. cinerea infection probably by inhibiting the activities of antioxidants and disease resistance enzymes, regulating SA and JA signaling pathways and modulating reactive oxygen (ROS) homeostasis.

Spermine and Spermidine Priming against Botrytis cinerea Modulates ROS Dynamics and Metabolism in Arabidopsis.

Polyamines (PAs) are ubiquitous small aliphatic polycations important for growth, development, and environmental stress responses in plants. Here, we demonstrate that exogenous application of spermine (Spm) and spermidine (Spd) induced cell death at high concentrations, but primed resistance against the necrotrophic fungus Botrytis cinerea in Arabidopsis. At low concentrations, Spm was more effective than Spd. Treatments with higher exogenous Spd and Spm concentrations resulted in a biphasic endogenous PA accumulation. Exogenous Spm induced the accumulation of H2O2 after treatment but also after infection with B. cinerea. Both Spm and Spd induced the activities of catalase, ascorbate peroxidase, and guaiacol peroxidase after treatment but also after infection with B. cinerea. The soluble sugars glucose, fructose, and sucrose accumulated after treatment with high concentrations of PAs, whereas only Spm induced sugar accumulation after infection. Total and active nitrate reductase (NR) activities were inhibited by Spm treatment, whereas Spd inhibited active NR at low concentrations but promoted active NR at high concentrations. Finally, γaminobutyric acid accumulated after treatment and infection in plants treated with high concentrations of Spm. Phenylalanine and asparagine also accumulated after infection in plants treated with a high concentration of Spm. Our data illustrate that Spm and Spd are effective in priming resistance against B. cinerea, opening the door for the development of sustainable alternatives for chemical pesticides.

The α-Subunit of the Chloroplast ATP Synthase of Tomato Reinforces Resistance to Gray Mold and Broad-Spectrum Resistance in Transgenic Tobacco.

Chloroplast ATP synthase (cpATPase) is responsible for ATP production during photosynthesis. Our previous studies showed that the cpATPase CF1 α subunit (AtpA) is a key protein involved in Clonostachys rosea-induced resistance to the fungus Botrytis cinerea in tomato. Here, we show that expression of the tomato atpA gene was upregulated by B. cinerea and Clonostachys rosea. The tomato atpA gene was then isolated, and transgenic tobacco lines were obtained. Compared with untransformed plants, atpA-overexpressing tobacco showed increased resistance to B. cinerea, characterized by reduced disease incidence, defense-associated hypersensitive response-like reactions, balanced reactive oxygen species, alleviated damage to the chloroplast ultrastructure of leaf cells, elevated levels of ATP content and cpATPase activity, and enhanced expression of genes related to carbon metabolism, photosynthesis, and defense. Incremental Ca2+ efflux and steady H+ efflux were observed in transgenic tobacco after inoculation with B. cinerea. In addition, overexpression of atpA conferred enhanced tolerance to salinity and resistance to the fungus Cladosporium fulvum. Thus, AtpA is a key regulator that links signaling to cellular redox homeostasis, ATP biosynthesis, and gene expression of resistance traits to modulate immunity to pathogen infection and provides broad-spectrum resistance in plants in the process.

Characterization of the Chitinase Gene Family in Mulberry (Morus notabilis) and MnChi18 Involved in Resistance to Botrytis cinerea.

Chitinase is a hydrolase that uses chitin as a substrate. It plays an important role in plant resistance to fungal pathogens by degrading chitin. Here, we conducted bioinformatics analysis and transcriptome data analysis of the mulberry (Morus notabilis) chitinase gene family to determine its role in the resistance to Botrytis cinerea. A total of 26 chitinase genes were identified, belonging to the GH18 and GH19 families. Among them, six chitinase genes were differentially expressed under the infection of B. cinerea. MnChi18, which significantly responded to B. cinerea, was heterologously expressed in Arabidopsis (Arabidopsis thaliana). The resistance of MnChi18 transgenic Arabidopsis to B. cinerea was significantly enhanced, and after inoculation with B. cinerea, the activity of catalase (CAT) increased and the content of malondialdehyde (MDA) decreased. This shows that overexpression of MnChi18 can protect cells from damage. In addition, our study also indicated that MnChi18 may be involved in B. cinerea resistance through other resistance-related genes. This study provides an important basis for further understanding the function of mulberry chitinase.

Phosphorylation of ATG18a by BAK1 suppresses autophagy and attenuates plant resistance against necrotrophic pathogens.

Autophagy is critical for plant defense against necrotrophic pathogens, which causes serious yield loss on crops. However, the post-translational regulatory mechanisms of autophagy pathway in plant resistance against necrotrophs remain poorly understood. In this study, we report that phosphorylation modification on ATG18a, a key regulator of autophagosome formation in Arabidopsis thaliana, constitutes a post-translation regulation of autophagy, which attenuates plant resistance against necrotrophic pathogens. We found that phosphorylation of ATG18a suppresses autophagosome formation and its subsequent delivery into the vacuole, which results in reduced autophagy activity and compromised plant resistance against Botrytis cinerea. In contrast, overexpression of ATG18a dephosphorylation-mimic form increases the accumulation of autophagosomes and complements the plant resistance of atg18a mutant against B. cinerea. Moreover, BAK1, a key regulator in plant resistance, was identified to physically interact with and phosphorylate ATG18a. Mutation of BAK1 blocks ATG18a phosphorylation at four of the five detected phosphorylation sites after B. cinerea infection and strongly activates autophagy, leading to enhanced resistance against B. cinerea. Collectively, the identification of functional phosphorylation sites on ATG18a and the corresponding kinase BAK1 unveiled how plant regulates autophagy during resistance against necrotrophic pathogens.Abbreviations:35s: the cauliflower mosaic virus 35s promoter; A. thaliana: Arabidopsis thaliana; A. brassicicola: Alternaria brassicicola; ABA: abscisic acid; ATG: autophagy-related; ATG18a: autophagy-related protein 18a in A. thaliana; ATG8a: autophagy-related protein 8a in A. thaliana; ATG8-PE: ATG8 conjugated with PE; B. cinerea: Botrytis cinerea; BAK1: Brassinosteroid insensitive 1-associated receptor kinase1 in A. thaliana; BiFC: biomolecular fluorescence complementation; BIK1: Botrytis-insensitive kinase 1 in A. thaliana; BKK1: BAK1-like 1 in A. thaliana; BR: brassinosteroid; Co-IP: coimmunoprecipitation; dai: days after inoculation; DAMPs: damage-associated molecular patterns; E. coli: Escherochia coli; ER: endoplasmic reticulum; ETI: effector-triggered immunity; GFP: green fluorescent protein; HA: hemagglutinin; IP: immunoprecipitation; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LCI: luciferase complementation imaging; MPK3: mitogen-activated protein kinase 3 in A. thaliana; MPK4: mitogen-activated protein kinase 4 in A. thaliana; MPK6: mitogen-activated protein kinase 6 in A. thaliana; N. benthamiana: Nicotiana benthamiana; NES: nuclear export sequence; PAMP: pathogen-associated molecular pattern; PCR: polymerase chain reaction; PE: phosphatidylethanolamine; PRR: pattern recognition receptor; PtdIns(3,5)P2: phosphatidylinositol (3,5)-biphosphate; PtdIns3P: phosphatidylinositol 3-biphosphate; PTI: PAMP-triggered immunity; qRT-PCR: quantitative reverse transcription PCR; SnRK2.6: SNF1-related protein kinase 2.6 in A. thaliana; TORC1: the rapamycin-sensitive Tor complex1; TRAF: tumor necrosis factor receptor-associated factor; WT: wild type plant; Yc: C-terminal fragment of YFP; YFP: yellow fluorescent protein; Yn: N-terminal fragment of YFP.