Induction Of Callose Deposition Research Articles

Theanine enhances resistance to Botrytis cinerea in postharvest strawberry fruit via modulating cell-wall and phenylpropanoid metabolisms

Theanine (N-ethyl-γ-glutamine), as a unique non-protein amino acid, plays vital roles in abiotic stress resistance, while its roles in biotic stress resistance are still unclear. Gray mold caused by Botrytis cinerea is a major disease in strawberries. Effects of theanine on the development of gray mold, cell-wall and phenylpropanoid metabolisms in strawberries were investigated in this study. Results showed that 5 mmol L−1 theanine treatment reduced disease incidence and severity of gray mold in strawberries with antifungal activity in vitro. Meanwhile, theanine treatment enhanced the accumulation of phenolic compounds and lignin, especially ellagic acid, cyanidin, and quercetin, which was associated with increased phenylpropanoid pathway related enzyme activities. Moreover, theanine induced callose deposition and suppressed cell- wall disassembling enzymes, accompanied by higher levels of water insoluble pectin, hemicellulose and cellulose. Therefore, theanine treatment could alleviate decay of B. cinerea-inoculated strawberries by regulating phenylpropanoid and cell-wall metabolisms, maintaining higher levels of phenolic compounds and cell-wall components, thereby contributing to disease resistance and cell-wall structure integrity.

Wheat Leaf Rust Fungus Effector Protein Pt1641 Is Avirulent to TcLr1.

Wheat leaf rust fungus is an obligate parasitic fungus that can absorb nutrients from its host plant through haustoria and secrete effector proteins into host cells. The effector proteins are crucial factors for pathogenesis as well as targets for host disease resistance protein recognition. Exploring the role of effector proteins in the pathogenic process of Puccinia triticina Eriks. (Pt) is of great significance for unraveling its pathogenic mechanisms. We previously found that a cysteine-rich effector protein, Pt1641, is highly expressed during the interaction between wheat and Pt, but its specific role in pathogenesis remains unclear. Therefore, this study employed techniques such as heterologous expression, qRT-PCR analysis, and host-induced gene silencing (HIGS) to investigate the role of Pt1641 in the pathogenic process of Pt. The results indicate that Pt1641 is an effector protein with a secretory function and can inhibit BAX-induced programmed cell death in Nicotiana benthamiana. qRT-PCR analyses showed that expression levels of Pt1641 were different during the interaction between the high-virulence strain THTT and low-virulence strains FGD and Thatcher, respectively. The highest expression level in the low-virulence strain FGD was four times that of the high-virulence strain THTT. The overexpression of Pt1641 in wheat near-isogenic line TcLr1 induced callose deposition and H2O2 production on TcLr1. After silencing Pt1641 in the Pt low-virulence strain FGD on wheat near-isogenic line TcLr1, the pathogenic phenotype of Pt physiological race FGD on TcLr1 changed from ";" to "3", indicating that Pt1641 plays a non-toxic function in the pathogenicity of FGD to TcLr1. This study helps to reveal the pathogenic mechanism of wheat leaf rust and provides important guidance for the mining and application of Pt avirulent genes.

FlgI Is a Sec-Dependent Effector of Candidatus Liberibacter asiaticus That Can Be Blocked by Small Molecules Identified Using a Yeast Screen.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas) is considered to be the main pathogen responsible for HLB. There is currently no effective practical strategy for the control of HLB. Our understanding of how pathogens cause HLB is limited because CLas has not been artificially cultured. In this study, 15 potential virulence factors were predicted from the proteome of CLas through DeepVF and PHI-base searches. One among them, FlgI, was found to inhibit yeast growth when expressed in Saccharomyces cerevisiae. The expression of the signal peptide of FlgI fused with PhoA in Escherichia coli resulted in the discovery that FlgI was a novel Sec-dependent secretory protein. We further found that the carboxyl-terminal HA-tagged FlgI was secreted via outer membrane vesicles in Sinorhizobium meliloti. Fluoresence localization of transient expression FlgI-GFP in Nicotiana benthamiana revealed that FlgI is mainly localized in the cytoplasm, cell periphery, and nuclear periphery of tobacco cells. In addition, our experimental results suggest that FlgI has a strong ability to induce callose deposition and cell necrosis in N. benthamiana. Finally, by screening a large library of compounds in a high-throughput format, we found that cyclosporin A restored the growth of FlgI-expressing yeast. These results confirm that FlgI is a novel Sec-dependent effector, enriching our understanding of CLas pathogenicity and helping to develop new and more effective strategies to manage HLB.

IAA-miR164a-NAC100L1 module mediates symbiotic incompatibility of cucumber/pumpkin grafted seedlings through regulating callose deposition.

Grafting is one of the key technologies to overcome the obstacles of continuous cropping, and improve crop yield and quality. However, the symbiotic incompatibility between rootstock and scion affects the normal growth and development of grafted seedlings after survival. The specific molecular regulation mechanism of graft incompatibility is still largely unclear. In this study, we found that the IAA-miR164a-NAC100L1 module induced callose deposition to mediate the symbiotic incompatibility of cucumber/pumpkin grafted seedlings. The incompatible combination (IG) grafting interface accumulated more callose, and the activity of callose synthase (CmCalS1) and IAA content were significantly higher than in the compatible combination (CG). Treatment with IAA polar transport inhibitor in the root of the IG plants decreased CmCalS activity and callose content. Furthermore, IAA negatively regulated the expression of Cm-miR164a, which directly targeted cleavage of CmNAC100L1. Interestingly, CmNAC100L1 interacted with CmCalS1 to regulate its activity. Further analysis showed that the interaction between CmNAC100L1 and CmCalS1 increased the activity of CmCalS1 in the IG plants but decreased it in the CG plants. Point mutation analysis revealed that threonine at the 57th position of CmCalS1 protein played a critical role to maintain its enzyme activity in the incompatible rootstock. Thus, IAA inhibited the expression of Cm-miR164a to elevate the expression of CmNAC100L1, which promoted CmNAC100L1 interaction with CmCalS1 to enhance CmCalS1 activity, resulting in callose deposition and symbiotic incompatibility of cucumber/pumpkin grafted seedlings.

Diversity and Traits of Multiple Biotic Stressors Elicit Differential Defense Responses in Legumes

In agroecosystems, plants frequently confront multiple biotic stressors, including herbivores and pathogens. The nature of these interactions plays a crucial role in mediating the activation of plant defense mechanisms. However, induction of plant chemical defenses has been more well studied than the induction of physical defenses. Here, we assessed the physical and chemical defense responses of pea (Pisum sativum) plants after exposure to three stressors: a vector herbivore (pea aphid, Acrythosiphon pisum), a non-vector herbivore (pea leaf weevil, Sitona lineatus), and a virus (Pea enation mosaic virus, PEMV). We used various histochemical staining techniques show that viruliferous A. pisum (transmitting PEMV) strongly induced callose deposition (aniline blue staining) and antioxidant-mediated defenses (DAB and NBT staining) in peas, primarily through accumulating reactive oxygen species (ROS). High-throughput phenotyping showed that viruliferous aphids reduced plant photosynthetic efficiency, but plants infected with PEMV had increased cell death (trypan blue staining). However, herbivory by aphids and weevils did not strongly induce defenses in peas, even though weevil feeding significantly reduced pea leaf area. These results show that not all herbivores induce strong defensive responses, and plant responses to vector species depends on their virus infection status. More broadly, our results indicate that variable stressors differentially regulate various plant responses through intricate chemical and physical defense pathways.

Induced defense response in soybean to Sclerotinia sclerotiorum using wuyiencin from Streptomyces albulus CK-15.

Sclerotinia sclerotiorum (Lib) de Bary, a destructive fungal pathogen with an extensive host range, causes major economic losses to crop production activities globally. Streptomyces spp. produce secondary metabolites with diverse structures and biological activities, with potential applications in the control of crop disease. This study explored the potential application of wuyiencin, a secondary metabolite of Streptomyces albulus CK-15, to induce defense responses in soybean against S. sclerotiorum. Lesion size was reduced by nearly 60%, in wuyiencin-treated soybean plants compared to plants infected with S. sclerotiorum only, in greenhouse experiments. Wuyiencin induced callose deposition at six hours postinoculation, and increase Reactive-Oxygen-Scavenging enzymes activities, including superoxide dismutase, catalase, and peroxidase activity. Moreover, wuyiencin inoculated before S. sclerotiorum infection significantly increased polyphenol oxidase, phenylalanine ammonia lyase, chitinase, and β-1,3-glucanase activity, suggesting their involvement in soybean defense responses to S. sclerotiorum. Further, qRT-PCR results showed expression levels of the hormone signaling markers CO11, MYC2, PR4, PR1, NPR1, and ERF1, were upregulated in infected leaves treated with wuyiencin.

The catalase-peroxidase PiCP1 plays a critical role in abiotic stress resistance, pathogenicity and asexual structure development in Phytophthora infestans.

Catalase-peroxidase is a heme oxidoreductase widely distributed in bacteria and lower eukaryotes. In this study, we identified a catalase-peroxidase PiCP1 (PITG_05579) in Phytophthora infestans. PiCP1 had catalase/peroxidase and secretion activities and was highly expressed in sporangia and upregulated in response to oxidative and heat stresses. Compared with wild type, PiCP1-silenced transformants (STs) had decreased catalase activity, reduced oxidant stress resistance and damped cell wall integrity. In contrast, PiCP1-overexpression transformants (OTs) demonstrated increased tolerance to abiotic stresses and induced the upregulation of PR genes in the host salicylic acid pathway. The high concentration of PiCP1 can also induced callose deposition in plant tissue. Importantly, both STs and OTs have severely reduced sporangia formation and zoospore releasing rate, but the sporangia germination rate and type varied depending on environmental conditions. Comparative sequence analyses show that catalase-peroxidases are broadly distributed and highly conserved among soil-borne plant parasitic oomycetes, but not in freshwater-inhabiting or strictly plants-inhabiting oomycetes. In addition, we found that silencing PiCP1 downregulated the expression of PiCAT2. These results revealed the important roles of PiCP1 in abiotic stress resistance, pathogenicity and in regulating asexual structure development in response to environmental change. Our findings provide new insights into catalase-peroxidase functions in eukaryotic pathogens.

Bacillus cereus EC9 protects tomato against Fusarium wilt through JA/ET-activated immunity.

The mechanisms of action and the limitations of effectiveness of natural biocontrol agents should be determined in order to convert them into end products that can be used in practice. Rhizosphere Bacillus spp. protect plants from various pathogens by displaying several modes of action. However, the ability of Bacillus spp. to control plant diseases depends on the interaction between the bacteria, host, and pathogen, and the environmental conditions. We found that soil drenching of tomato plants with the non-antifungal Bacillus cereus strain EC9 (EC9) enhances plant defense against Fusarium oxysporum f. sp. lycopersici (Fol). To study the involvement of plant defense-related phytohormones in the regulation of EC9-activated protection against Fol, we conducted plant bioassays in tomato genotypes impaired in salicylic acid (SA) accumulation, jasmonic acid (JA) biosynthesis, and ethylene (ET) production, and analyzed the transcript levels of pathways-related marker genes. Our results indicate that JA/ET-dependent signaling is required for EC9-mediated protection against Fol in tomato. We provide evidence that EC9 primes tomato plants for enhanced expression of proteinase inhibitor I (PI-I) and ethylene receptor4 (ETR4). Moreover, we demonstrated that EC9 induces callose deposition in tomato roots. Understanding the involvement of defense-related phytohormones in EC9-mediated defense against Fusarium wilt has increased our knowledge of interactions between non-antifungal plant defense-inducing rhizobacteria and plants.

Cytokinin Deficiency Alters Leaf Proteome and Metabolome during Effector-Triggered Immunity in Arabidopsis thaliana Plants.

The involvement of cytokinins (CK) in biotic stresses has been recognized, while knowledge regarding the effects of CK deficiency on plant response against pathogens is less abundant. Thus, the purpose of this study was to reveal the effects of CK deficiency on proteomics and metabolomic responses of flg22-triggered immunity. We conducted a series of histochemical assays to investigate the activity of the downstream pathways caused by flg22, such as accumulation of ROS, induction of defence genes, and callose deposition, that occurred in Arabidopsis thaliana transgenic lines overexpressing the Hordeum vulgare CKX2 gene (HvCKX2), which are therefore CK-deficient. We also used GC and LC-MS-based technology to quantify variations in stress hormone levels and metabolomic and proteomic responses in flg22-treated HvCKX2 and wild-type Arabidopsis plants. We found that CK deficiency alters the flg22-triggered plant defence response, especially through induction of callose deposition, upregulation of defence response-related proteins, increased amino acid biosynthesis, and regulation of plant photosynthesis. We also indicated that JA might be an important contributor to immune response in plants deficient in CKs. The present study offers new evidence on the fundamental role of endogenous CK in the response to pathogens, as well as the possibility of altering plant biotic tolerance by manipulating CK pools.

Roles of the EPS66A polysaccharide from Streptomyces sp. in inducing tobacco resistance to tobacco mosaic virus

EPS66A was derived from an unidentified Streptomyces sp. HL-66 by chemical fraction and disease-resistance assays. It was identified as a polysaccharide through a series of chemical characterization, including infrared spectrum analysis, methylation, gas chromatography–mass spectrometry, nuclear magnetic resonance, and high-performance gel permeation chromatography. To determine its effect in plant, EPS66A was applied to tobacco leaves infected with TMV, resulting in the plant with enhanced systemic resistance with a significant reduction of TMV severity. Plant defense was confirmed by early responses, including hypersensitive response (HR) indicated by programed cell death, moderate alkalization, oxidative burst, increase in nitric oxide (NO) and salicylic acid (SA). Furthermore, EPS66A induced callose deposition to form defense barriers against pathogen invasion and the expression of pathogenesis-related (PR) genes, which confirmed the second level of plant defense. Therefore, EPS66A served as a resistance inducer, which was reorganized by tobacco cells that triggered the production of signal molecules. The signals moved in long distance and systemically in plant, which coordinated the expression of defense responses. The study provided a new perspective in understanding the mechanism of EPS66A in regulating plants on environmental adaptability and provided a theoretical foundation for designing safe and sustainable pesticides.

Chitosan nanoparticles mitigate Alternaria leaf spot disease of chilli in nitric oxide dependent way

The present study focuses on protection of emerging Alternaria leaf spot disease of chilli by application of chitosan nanoparticles (CNP). CNP was prepared by ionotropic gelation method and characterized. Antifungal potential of CNP was also checked against Alternaria alternata and its mechanisms were unraveled. Foliar application of CNP (0.001%) improved plant innate immunity in two chilli cultivars (one tolerant and one susceptible) by inducing the activities of different defense related enzymes along with total phenol and flavonoid. CNP application also induced callose deposition and reduced cell death in both the cultivar. Signaling molecule nitric oxide (NO) also augmented in CNP treated sets which were confirmed by both biochemical and microscopic data. In order to find out involvement of NO in CNP induced innate immunity in chilli cultivars, both NO surplus and NO depleted conditions were artificially created and defense responses were recorded. It was interesting to note that CNP mediated enhancement of defense responses in chilli plants was compromised in NO depleted condition. These results signify possible involvement of NO in CNP induced defense responses in chilli plants. It is evident from our results that CNP can be used to protect chilli plants against this fungal disease to develop a sustainable management strategy.

Activating the MYB51 and MYB122 to upregulate the transcription of glucosinolates biosynthesis genes by copper ions in Arabidopsis

Copper ions (Cu2+) are key constituents of copper-based antimicrobial compounds (CBACs), which are extensively used in agriculture. Previously, we demonstrated that a low concentration of Cu2+ induced plant defenses associated with callose deposition in Arabidopsis as well as flg22, a microbe-associated molecular pattern (MAMP) peptide. However, the details and differences of the mechanisms between Cu2+- and flg22-mediated callose deposition remain unclear. Here, we reported that Cu2+- and flg22-induced defense responses and callose deposition are dependent on AtACS8 and AtACS2/AtACS6, respectively. After the RNA sequencing data were mined, the expression of MYB51, MYB122, CYP79B2/B3 and CYP83B1 implied that a conserved downstream indole glucosinolate (IGS) metabolic pathway is regulated by Cu2+. In the Cu2+-induced response, the ethylene biosynthesis rate-limiting gene AtACS8 and the signal transduction pathway were found to be required for Cu2+-activated MYB51 and MYB122 transcription. Functional redundancy of MYB51 and MYB122, the key regulators of the IGS metabolic pathway, was identified in the Cu2+-mediated regulation of IGS gene transcription, promotion of callose deposition, and increase in Arabidopsis resistance to bacterial pathogens. Furthermore, IGS genes such as CYP79B2, CYP81F2 and PAD2 were required for Cu2+-induced callose deposition and defense responses. Our results demonstrate that Cu2+ activates MYB51 and MYB122 through distinct ethylene signal transduction to regulate the IGS metabolic pathway, resulting in an enhanced defense response in Arabidopsis.

Treatment of Penicillium chrysogenum extracts (PDMP) restricts the spread of Tobacco mosaic virus by priming callose deposition in Nicotiana benthamiana

Plants are often primed to enhanced resistance upon recognizing certain microbes and microbial metabolites/molecules. We investigated the effect of PDMP, the peptides extract of dry mycelia of Penicillium chrysogenum, on defense of Nicotiana benthamiana to Tobacco mosaic virus (TMV). The 30B vector harboring the green fluorescent protein gene (30B:GFP) was used to monitor virus movement. Movement of the virus vector was limitied in PDMP-pretreated tobacco, in which significant callose accumulation was observed after challenge inoculation with the TMV vector. Callose accumulation was absent in plants pretreated with both the PDMP and callose synthesis inhibitor, resulting in elimination of PDMP-induced inhibition of TMV spread. Transcripts of the defense-related genes were significantly accumulated after challenge inoculation with TMV in PDMP-pretreated tobacco. Interestingly, PDMP treatment or TMV inoculation alone did neither induce callose deposition, nor enhance the transcript abundance of the defense-related genes, indicating that PDMP was more likely to promote tobacco to a primed state than induce defense responses directly. The primed callose accumulation plays a crucial role in restricting TMV spread in PDMP-pretreated tobacco. The present results provide evidence of PDMP can promote N. benthamiana to a primed state of enhance defense against TMV.

A novel glycoprotein from Streptomyces sp. triggers early responses of plant defense

GP-1, a novel glycoprotein from Streptomyces sp. ZX01 has a plant immunity-inducing effect. GP-1-treated plants exhibited enhanced systemic resistance with a significant reduction in TMV lesions on tobacco leaves, but its antiviral mechanism remains unclear. In this study, early plant defense-related responses, such as Ca2+ influx, callose apposition, oxidative burst, hypersensitive response, programmed cell death, increase in nitric oxide (NO), and stomatal closure, were investigated under GP-1 treatment, and the mechanism of how GP-1 induces viral resistance in Nicotiana benthamiana was studied. Results showed that GP-1 induced [Ca2+]cyt rapidly in tobacco leaves and suspended cells, followed by reactive oxygen species (ROS) and NO elevation. Transcriptome analysis showed significant differences in expression levels between the GP-1-treated N. benthamiana and the control and showed significantly upregulated and enriched pathways including defense and immune reaction. Similar to typical pathogen-associated molecular patterns, GP-1 induced callose deposition and stomatal closure to form defense barriers against pathogen invasion. The expression of defense-related genes further confirmed the above conclusions. By analyzing transcriptome in N. benthamiana and the contents of salicylic acid (SA) and jasmonic acid (JA), GP-1 enhanced viral resistance of tobacco by improving the SA and JA contents, strengthening plant secondary metabolites activities, promoting systemic accumulation of pathogenesis-related proteins in TMV- inoculated tobacco there by producing antiviral activity.

Crop Protection against Botrytis cinerea by Rhizhosphere Biological Control Agent Bacillus velezensis XT1.

This study aims to evaluate the use of Bacillus velezensis strain XT1 as a plant growth-promoting rhizobacterium (PGPR) and biocontrol agent against B. cinerea in tomato and strawberry plants. Foliar and radicular applications of strain XT1 increased plant total biomass as compared to the control and B. cinerea-infected plants, with root applications being, on the whole, the most effective mode of treatment. Applications of the bacterium were found to reduce infection parameters such as disease incidence and severity by 50% and 60%, respectively. We analyzed stress parameters and phytohormone content in order to evaluate the capacity of XT1 to activate the defense system through phytohormonal regulation. Overall, the application of XT1 reduced oxidative damage, while the H2O2 and malondialdehyde (MDA) content was lower in XT1-treated and B. cinerea-infected plants as compared to non-XT1-treated plants. Moreover, treatment with XT1 induced callose deposition, thus boosting the response to pathogenic infection. The results of this study suggest that the signaling and activation pathways involved in defense mechanisms are mediated by jasmonic acid (JA) and ethylene hormones, which are induced by preventive treatment with XT1. The study also highlights the potential of preventive applications of strain XT1 to activate defense mechanisms in strawberry and tomato plants through hormone regulation.

Salmonella enterica Serovar Typhimurium 14028s Genomic Regions Required for Colonization of Lettuce Leaves.

Contamination of edible produce leaves with human bacterial pathogens has been associated with serious disease outbreaks and has become a major public health concern affecting all aspects of the market, from farmers to consumers. While pathogen populations residing on the surface of ready-to-eat produce can be potentially removed through thorough washing, there is no disinfection technology available that effectively eliminates internal bacterial populations. By screening 303 multi-gene deletion (MGD) mutants of Salmonella enterica serovar Typhimurium (STm) 14028s, we were able to identify ten genomic regions that play a role in opening the stomatal pore of lettuce leaves. The major metabolic functions of the deleted regions are associated with sensing the environment, bacterium movement, transport through the bacterial membrane, and biosynthesis of surface appendages. Interestingly, at 21 days post inoculation, seven of these mutants showed increased population titers inside the leaf, two mutants showed similar titers as the wild type bacterium, whereas one mutant with a large deletion that includes the Salmonella pathogenicity island 2 (SPI-2) showed significantly impaired persistence in the leaf apoplast. These findings suggest that not all the genomic regions required for initiation of leaf colonization (i.e., epiphytic behavior and tissue penetration) are essential for continuing bacterial survival as an endophyte. We also observed that mutants lacking either SPI-1 (Mut3) or SPI-2 (Mut9) induce callose deposition levels comparable to those of the wild type STm 14028s; therefore, these islands do not seem to affect this lettuce defense mechanism. However, the growth of Mut9, but not Mut3, was significantly impaired in the leaf apoplastic wash fluid (AWF) suggesting that the STm persistence in the apoplast may be linked to nutrient acquisition capabilities or overall bacterial fitness in this niche, which are dependent on the gene(s) deleted in the Mut9 strain. The genetic basis of STm colonization of leaves investigated in this study provides a foundation from which to develop mitigation tactics to enhance food safety.

Brassinosteroids are involved in ethylene-induced Pst DC3000 resistance in Nicotiana benthamiana.

Plant immunity is regulated by a huge phytohormone regulation network. Ethylene(ET) and brassinosteroids (BRs) play critical roles in plant response to biotic stress; however, the relationship between BR and ET in plant immunity is unclear. We used chemical treatments, genetic approaches and inoculation experiments to investigate the relationship between ET and BR in plant defense against Pst DC3000 in Nicotiana benthamiana. Foliar applications of ET and BR enhanced plant resistance to Pst DC3000 inoculation, while treatment with brassinazole (BRZ, a specific BR biosynthesis inhibitor) eliminated the ET induced plant resistance to Pst DC3000. Silencing of DWARF 4(DWF4, a key BR biosynthetic gene), BRASSINOSTEROID INSENSITIVE 1 (BRI1, aBR receptor) and BRASSINOSTEROID-SIGNALING KINASE 1 (BSK1, downstream of BRI1) also neutralised the ET-induced plant resistance to Pst DC3000. ET can induce callose deposition and reactive oxygen species (ROS) accumulation to resistPst DC3000, BRZ-treated and gene-silenced were completely eliminate this response. Our results suggest BR is involved in ET-induced plant resistance, the involvement of ET in plant resistance is possibly by the induction of callose deposition and ROS accumulation, in a BR-dependent manner.

Overexpression of OsPUB41, a Rice E3 ubiquitin ligase induced by cell wall degrading enzymes, enhances immune responses in Rice and Arabidopsis

BackgroundCell wall degrading enzymes (CWDEs) induce plant immune responses and E3 ubiquitin ligases are known to play important roles in regulating plant defenses. Expression of the rice E3 ubiquitin ligase, OsPUB41, is enhanced upon treatment of leaves with Xanthomonas oryzae pv. oryzae (Xoo) secreted CWDEs such as Cellulase and Lipase/Esterase. However, it is not reported to have a role in elicitation of immune responses.ResultsExpression of the rice E3 ubiquitin ligase, OsPUB41, is induced when rice leaves are treated with either CWDEs, pathogen associated molecular patterns (PAMPs), damage associated molecular patterns (DAMPs) or pathogens. Overexpression of OsPUB41 leads to induction of callose deposition, enhanced tolerance to Xoo and Rhizoctonia solani infection in rice and Arabidopsis respectively. In rice, transient overexpression of OsPUB41 leads to enhanced expression of PR genes and SA as well as JA biosynthetic and response genes. However, in Arabidopsis, ectopic expression of OsPUB41 results in upregulation of only JA biosynthetic and response genes. Transient overexpression of either of the two biochemically inactive mutants (OsPUB41C40A and OsPUB41V51R) of OsPUB41 in rice and stable transgenics in Arabidopsis ectopically expressing OsPUB41C40A failed to elicit immune responses. This indicates that the E3 ligase activity of OsPUB41 protein is essential for induction of plant defense responses.ConclusionThe results presented here suggest that OsPUB41 is possibly involved in elicitation of CWDE triggered immune responses in rice.

Interplay between phosphoinositides and actin cytoskeleton in the regulation of immunity related responses in Arabidopsis thaliana seedlings

Actin cytoskeleton is indispensable for plant cell integrity. Besides, increasing evidences illustrate its crucial role in plant responses to environment, including defence against pathogens. Recently, we have demonstrated that pre-treatment with actin disrupting drugs latrunculin B (latB) and cytochalasin E can enhance plant resistance against bacterial and fungal pathogens via activation of salicylic acid (SA) pathway. Here, we show that actin depolymerization in Arabidopsis thaliana seedlings not only triggers SA biosynthesis by ICS1, but also induces callose deposition via callose synthase PMR4. This effect is SA-independent since still present in mutants that do not accumulate SA. LatB also triggers the expression of several defence related genes. We could distinguish genes, induced in a SA-dependent manner (PR1, WRKY38, ICS1) and those that are SA-independent (PR2, PAD4, BAP1). As actin cytoskeleton is tightly connected with membrane trafficking, we assayed the effect of latB on mutant plants invalidated in phosphatidylinositol 4-kinase beta1 and beta2 (PI4Kβ1β2). Deficiency in PI4Kβ1β2 enhanced latB-triggered actin filaments depolymerisation. Yet, it did not lead to a stronger callose deposition or SA biosynthesis in response to latB. Surprisingly, introduction of NahG construct or pad4 mutation in pi4kß1ß2 background had much lower effect on SA induction and SA-dependent gene expression changes than it has in wild type. We can thus conclude that actin disruption itself triggers immune-like responses: there is an induction of SA via PAD4 coupled to ICS1; it leads to the induction of PR1 and WRKY38, and this requires a functional PI4Kβ1β2 to be properly regulated. However, an alternative, SA-independent pathway also exists that leads to the enhanced expression of PR2 and to callose accumulation.

A substrate of the ABC transporter PEN3 stimulates bacterial flagellin (flg22)-induced callose deposition in Arabidopsis thaliana

Nonhost resistance of Arabidopsis thaliana against Phytophthora infestans, a filamentous eukaryotic microbe and the causal agent of potato late blight, is based on a multilayered defense system. Arabidopsis thaliana controls pathogen entry through the penetration-resistance genes PEN2 and PEN3, encoding an atypical myrosinase and an ABC transporter, respectively, required for synthesis and export of unknown indole compounds. To identify pathogen-elicited leaf surface metabolites and further unravel nonhost resistance in Arabidopsis, we performed untargeted metabolite profiling by incubating a P. infestans zoospore suspension on leaves of WT or pen3 mutant Arabidopsis plants. Among the plant-secreted metabolites, 4-methoxyindol-3-yl-methanol and S-(4-methoxy-indol-3-yl-methyl) cysteine were detected in spore suspensions recollected from WT plants, but at reduced levels from the pen3 mutant plants. In both whole-cell and microsome-based assays, 4-methoxyindol-3-yl-methanol was transported in a PEN3-dependent manner, suggesting that this compound is a PEN3 substrate. The syntheses of both compounds were dependent on functional PEN2 and phytochelatin synthase 1. None of these compounds inhibited mycelial growth of P. infestans in vitro Of note, exogenous application of 4-methoxyindol-3-yl methanol slightly elevated cytosolic Ca2+ levels and enhanced callose deposition in hydathodes of seedlings treated with a bacterial pathogen-associated molecular pattern (PAMP), flagellin (flg22). Loss of flg22-induced callose deposition in leaves of pen3 seedlings was partially reverted by the addition of 4-methoxyindol-3-yl methanol. In conclusion, we have identified a specific indole compound that is a substrate for PEN3 and contributes to the plant defense response against microbial pathogens.