Dual RNA-seq reveals distinct families of co-regulated and structurally conserved effectors in Botrytis cinerea infection of Arabidopsis thaliana.

BACKGROUND: Botrytis cinerea is a broad-host-range pathogen causing gray mold disease and significant yield losses of numerous crops. However, the mechanisms underlying its rapid invasion and efficient killing of plant cells remain unclear. RESULTS: In this study, we elucidated the dynamics of B. cinerea infection in Arabidopsis thaliana by live cell imaging and dual RNA sequencing. We found extensive transcriptional reprogramming events in both the pathogen and the host, which involved metabolic pathways, signaling cascades, and transcriptional regulation. For the pathogen, we identified 591 candidate effector proteins (CEPs) and comprehensively analyzed their co-expression, sequence similarity, and structural conservation. The results revealed temporal co-regulation patterns of these CEPs, indicating coordinated deployment of effectors during B. cinerea infection. Through functional screening of 48 selected CEPs in Nicotiana benthamiana, we identified 11 cell death-inducing proteins (CDIPs) in B. cinerea. CONCLUSIONS: The findings provide important insights into the transcriptional dynamics and effector biology driving B. cinerea pathogenesis. The rapid infection of this pathogen involves the temporal co-regulation of CEPs and the prominent role of CDIPs in host cell death. This work highlights significant changes in gene expression associated with gray mold disease, underscoring the importance of a diverse repertoire of effectors crucial for successful infection.

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.

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.

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.

Arabidopsis ABCG34 contributes to defense against necrotrophic pathogens by mediating the secretion of camalexin.

Plant pathogens cause huge yield losses. Plant defense often depends on toxic secondary metabolites that inhibit pathogen growth. Because most secondary metabolites are also toxic to the plant, specific transporters are needed to deliver them to the pathogens. To identify the transporters that function in plant defense, we screened Arabidopsis thaliana mutants of full-size ABCG transporters for hypersensitivity to sclareol, an antifungal compound. We found that atabcg34 mutants were hypersensitive to sclareol and to the necrotrophic fungi Alternaria brassicicola and Botrytis cinereaAtABCG34 expression was induced by Abrassicicola inoculation as well as by methyl-jasmonate, a defense-related phytohormone, and AtABCG34 was polarly localized at the external face of the plasma membrane of epidermal cells of leaves and roots. atabcg34 mutants secreted less camalexin, a major phytoalexin in Athaliana, whereas plants overexpressing AtABCG34 secreted more camalexin to the leaf surface and were more resistant to the pathogen. When treated with exogenous camalexin, atabcg34 mutants exhibited hypersensitivity, whereas BY2 cells expressing AtABCG34 exhibited improved resistance. Analyses of natural Arabidopsis accessions revealed that AtABCG34 contributes to the disease resistance in naturally occurring genetic variants, albeit to a small extent. Together, our data suggest that AtABCG34 mediates camalexin secretion to the leaf surface and thereby prevents Abrassicicola infection.

eIF2α Phosphorylation by GCN2 Is Induced in the Presence of Chitin and Plays an Important Role in Plant Defense against B. cinerea Infection.

Translation plays an important role in plant adaptation to different abiotic and biotic stresses; however, the mechanisms involved in translational regulation during each specific response and their effect in translation are poorly understood in plants. In this work, we show that GCN2 promotes eIF2α phosphorylation upon contact with Botrytis cinerea spores, and that this phosphorylation is required for the proper establishment of plant defense against the fungus. In fact, independent gcn2 mutants display an enhanced susceptibility to B. cinerea infection, which is highlighted by an increased cell death and reduced expression of ethylene- and jasmonic-related genes in the gcn2 mutants. eIF2α phosphorylation is not only triggered in the presence of the fungus, but interestingly, is also achieved in the sole presence of the microbe-associated molecular pattern (MAMP) chitin. Moreover, analysis of de novo protein synthesis by 35SMet-35SCys incorporation indicates that chitin treatment promotes a global inhibition of translation. Taken together, these results suggest that eIF2α phosphorylation by GCN2 is promoted in the presence of chitin and plays an important role in plant defense against B. cinerea infection.

Verifying the biocontrol activity of novel film-forming formulations of Candida sake CPA-1: resilience in relation to environmental factors, rainfall episodes, and control of Botrytis cinerea on different hosts.

BACKGROUND: The efficacy of Candida sake CPA-1 as a biocontrol agent against several diseases has been studied since it was isolated 20 years ago. However, it was only recently that two suitable and effective film-forming formulations based on potato starch and maltodextrins were developed using the fluidized-bed spray-drying system. The present work aimed to confirm the capability of both novel formulations by testing their resilience on grapes at different temperatures (0, 22, and 30 °C), relative humidities (40% and 85%), and simulated rainfall levels. Another objective was to examine the control of Botrytis cinerea in different hosts. RESULTS: The CPA-1 cells from both dried formulations survived better than the liquid formulation on grapes stored at 0 and 22 °C regardless of the relative humidity. After simulated rainfall, potato starch formulation achieved significantly higher populations than maltodextrin formulation, although the highest reduction was -1.6 log N N0 -1 . A positive effect of cell establishment prior to the simulated rainfall was shown, and recovered cells from the potato starch formulation were significantly higher after 72 h of cell establishment. Finally, both formulations reduced the incidence and severity of B. cinerea on pears, apples, and tomatoes. CONCLUSION: The potential of these novel film-forming formulations of C. sake CPA-1 was verified. The resilience of formulated C. sake was better than the commercialized liquid formulation, the adherence of the formulations to the grapes improved after an establishment period prior to rain exposure, and the control of B. cinerea was verified in a wider range of hosts. © 2019 Society of Chemical Industry.

Effect of ozonation on table grapes preservation in cold storage.

Table grapes artificially inoculated with B. Cinerea were tested under four different ozonation strategies in order to achieve prolongation of table grapes' shelf-life time. Decay incidence, external disease appearance, number of infected grapes, weight loss and a variety of quality parameters such as sugars and proteins content were checked after every 3 days. No significant alteration of table grapes quality characteristics was observed after their exposure to ozone atmosphere. Moreover, the low ozone dosage process (0.3 ppm) caused sufficient restriction of fruit decay due to fungal contamination and secured a 40-days-period of storage time. However, the observed weight loss was somewhat higher on the treated samples compared to the untreated ones. From technical and economical point of view the low dosage (0.3 ppm) ozonation process on a daily basis combined with the cold storage appears to a very promising method for table grapes preservation.

The role of FaBG3 in fruit ripening and B. cinerea fungal infection of strawberry.

In plants, ß-glucosidases (BG) have been implicated in developmental and pathogen defense, and are thought to take part in abscisic acid (ABA) synthesis via hydrolysis of ABA glucose ester to release active ABA; however, there is no genetic evidence for the role of BG genes in ripening and biotic/abiotic stress in fruits. To clarify the role of BG genes in fruit, eight Fa/FvBG genes encoding ß-glucosidase were isolated using information from the GenBank strawberry nucleotide database. Of the Fa/FvBG genes examined, expression of FaBG3 was the highest, showing peaks at the mature stage, coincident with the changes observed in ABA content. To verify the role of this gene, we suppressed the expression of FaBG3 via inoculation with Agrobacterium tumefaciens containing tobacco rattle virus carrying a FaBG3 fragment (RNAi). The expression of FaBG3 in FaBG3-RNAi-treated fruit was markedly reduced, and the ABA content was lower than that of the control. FaBG3-RNAi-treated fruit did not exhibit full ripening, and were firmer, had lower sugar content, and were pale compared with the control due to down-regulation of ripening-related genes. FaBG3-RNAi-treated fruit with reduced ABA levels were much more resistant to Botrytis cinerea fungus but were more sensitive to dehydration stress than control fruit. These results indicate that FaBG3 may play key roles in fruit ripening, dehydration stress and B. cinerea fungal infection in strawberries via modulation of ABA homeostasis and transcriptional regulation of ripening-related genes.

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.

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.

The Fungal Transcription Factor BcTbs1 from Botrytis cinerea Promotes Pathogenicity via Host Cellulose Degradation.

Zn(II)2Cys6 proteins constitute the largest group of fungal-specific transcription factors. However, little is known about their functions in the crop killer Botrytis cinerea. In this work, a T-DNA insertion strain M13448 was identified which was inserted into the Zn(II)2Cys6 TF-encoding gene BcTBS1. Knockout of BcTBS1 did not affect mycelia growth, appressorium formation, and sclerotium germination, but impaired fungal conidiation, conidial morphogenesis, conidial germination, infection cushion development, and sclerotial formation. Accordingly, ΔBctbs1 mutants showed reduced virulence in its host plants. Further study proved that BcTBS1, BCIN_15g03870, and BCIN_12g06630 were induced by cellulose. Subsequent cellulase activity assays revealed that the loss of BcTBS1 significantly decreased cellulase activity. In addition, we verified that the BCIN_15g03870 and BCIN_12g06630 genes were positive regulated by BcTBS1 by quantitative real-time reverse-transcription-polymerase chain reaction (qRT-PCR). Taken together, these results suggested that BcTBS1 can promote pathogenicity by modulating cellulase-encoding genes that participate in host cellulose degradation.

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.

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.

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.

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.

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.

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.