Protein Elicitor Research Articles

A novel protein elicitor (Cs08297) from Ciboria shiraiana enhances plant disease resistance.

Ciboria shiraiana is a necrotrophic fungus that causes mulberry sclerotinia disease resulting in huge economic losses in agriculture. During infection, the fungus uses immunity elicitors to induce plant tissue necrosis that could facilitate its colonization on plants. However, the key elicitors and immune mechanisms remain unclear in C. shiraiana. Herein, a novel elicitor Cs08297 secreted by C. shiraiana was identified, and it was found to target the apoplast in plants to induce cell death. Cs08297 is a cysteine-rich protein unique to C. shiraiana, and cysteine residues in Cs08297 were crucial for its ability to induce cell death. Cs08297 induced a series of defence responses in Nicotiana benthamiana, including the burst of reactive oxygen species (ROS), callose deposition, and activation of defence-related genes. Cs08297 induced-cell death was mediated by leucine-rich repeat (LRR) receptor-like kinases BAK1 and SOBIR1. Purified His-tagged Cs08297-thioredoxin fusion protein triggered cell death in different plants and enhanced plant resistance to diseases. Cs08297 was necessary for sclerotial development, oxidative-stress adaptation, and cell wall integrity but negatively regulated virulence of C. shiraiana. In conclusion, our results revealed that Cs08297 is a novel fungal elicitor in fungi inducing plant immunity. Furthermore, its potential to enhance plant resistance provides a new target to control agricultural diseases biologically.

Unveiling protein elicitor MoHrip2: A potent biocontrol agent extracted from Magnaporthe oryzae safeguards Australian tobacco (Nicotiana benthamiana) against tobacco aphid (Myzus nicotianae)

The tobacco aphid (Myzus nicotianae) poses a significant threat to global Australian tobacco (Nicotiana benthamiana) output by inflicting damage through leaf twisting and feeding during its early growth stages. To explore potential herbivore resistance in N. benthamiana, this study investigates the effects of the elicitor protein MoHrip2, derived from Magnaporthe oryzae, on the immature growth, survival, and lifespan of M. nicotianae. Compared to the control group, MoHrip2 treatments at three concentrations (85.24, 51.14, 25.57 μg/mL) reduced M. nicotianae population after 7 days, 14 days, and 21 days. All concentrations significantly reduced aphid colonization rate, except for the lowest elicitor concertation (25.57 μg/mL at 7D). Likewise compared to the control group, the highest concentration of MoHrip2 i.e. 85.24 μg/mL, resulted in a decrease in the growth rates of M. nicotianae (second and third-generation). M. nicotianae colonized control plants quicker than MoHrip2-treated N. benthamiana plants in a host selection test. MoHrip2 (85.24, 51.14, 25.57 μg/mL) concentrations extended M. nicotianae nymphal development. The trichomes and wax on MoHrip2-treated leaves made M. nicotianae environment hostile. M. nicotianae reproduction and colonization were reduced by MoHrip2's leaf surface changes. Electrical penetration graphs (EPGs) against M. nicotianae assessed adult reproductive efficiency and pest-pathogen interaction. MoHrip2-treated N. benthamiana seedlings exhibited increased amounts of expression of genes involved in jasmonic acid (JA), salicylic acid (SA) pathways indicating the activation of systemic defense against M. nicotianae. This study reveals a direct correlation between temperature variations and climatic shifts in N. benthamiana plants, highlighting the potential of MoHrip2 in revolutionizing agroecosystems through enhanced pest management and biocontrol strategies. In conclusion, the present study sheds new light on the potential of MoHrip2, derived from M. oryzae, as an effective biocontrol technique against M. nicotianae, infesting N. benthamiana. These findings provide valuable insights into developing innovative and sustainable solutions for pest management in agricultural practices, considering the evidence presented against M. nicotianae, it can be concluded that implementing integrated pest management and utilizing MoHrip2 for bio control in the agroecosystem is an appropriate approach.

Characterisation and expression analyses of two putative cerato-platanin proteins isolated from Ganoderma boninense Pat.

Cerato-platanin (CP) proteins are small extracellular proteins that play essential roles in the plant-pathogen interactions of some economically important filamentous fungi. At present, there is a lack of information about the roles of CP proteins secreted by Ganoderma boninense, the causal agent of basal stem rot disease in African oil palm, during the early host-pathogen interaction. This work studies the properties and roles of two CP proteins from G. boninense strain PER71. Using bioassay-guided fractionation and nano-LC-MS/MS analysis, we identified peptide matches for G. sinense ZZ0214-1 CP-like and other elicitor proteins in the culture filtrate of G. boninense strain PER71. We found that GbCP2 and GbCP6, two out of nine CP gene models identified in the fungal genome of G. boninense strain PER71, exhibited potential effector properties. We further validated their effector properties by isolating and characterising their coding regions. Interestingly, GbCP6 showed a significant expression level at 11 days post inoculation, indicating its significance in fungal pathogenesis. On the other hand, GbCP2 did not show differential expression at any sampling point, suggesting a different function or role in the pathogenicity of G. boninense. This study identifies GbCP6 as a potential biomarker for early detection of basal stem rot disease in oil palm.

Joint application of plant immunity-inducing elicitors and fungicides to control Phytophthora diseases

Phytophthora are destructive plant pathogens that pose a serious threat to crop production. Traditional control methods rely heavily on chemical fungicides, which are harmful to the environment and human health. Currently, effective green prevention and control methods for Phytophthora pathogens are lacking. Plants rely primarily on their innate immune system to resist pathogens. Plant cells perceive pathogen invasion and activate immune responses by recognizing specific pathogen-derived molecules, called elicitors, which mainly include pathogen-associated molecular patterns (PAMPs) and microbial effector proteins. PAMPs, which are conserved molecular features of microbes and recognized by plant cell surface-localized pattern-recognition receptors (PRRs), activate mild and broad-spectrum disease resistance. However, there are few reports on elicitor proteins that induce broad resistance against Phytophthora pathogens. In this study, we identified BcIEB1, a fungal-derived PAMP, which activated plant immune responses in a BAK1- and SOBIR1-dependent manner. BcIEB1 could induce plant resistance to various Phytophthora pathogens, including P. capsici, P. infestans, and P. parasitica. We further found that the combination of lower concentrations of BcIEB1 with fungicides, such as pyraclostrobin, azoxystrobin, and metalaxyl-M, could enhance the effect on Phytophthora disease control while reducing the dependence on fungicides, thereby reducing environmental pollution. This study identified a novel, less toxic strategy for controlling Phytophthora diseases.

Rice false smut virulence protein subverts host chitin perception and signaling at lemma and palea for floral infection.

The flower-infecting fungus Ustilaginoidea virens causes rice false smut, which is a severe emerging disease threatening rice (Oryza sativa) production worldwide. False smut not only reduces yield, but more importantly produces toxins on grains, posing a great threat to food safety. U. virens invades spikelets via the gap between the 2 bracts (lemma and palea) enclosing the floret and specifically infects the stamen and pistil. Molecular mechanisms for the U. virens-rice interaction are largely unknown. Here, we demonstrate that rice flowers predominantly employ chitin-triggered immunity against U. virens in the lemma and palea, rather than in the stamen and pistil. We identify a crucial U. virens virulence factor, named UvGH18.1, which carries glycoside hydrolase activity. Mechanistically, UvGH18.1 functions by binding to and hydrolyzing immune elicitor chitin and interacting with the chitin receptor CHITIN ELICITOR BINDING PROTEIN (OsCEBiP) and co-receptor CHITIN ELICITOR RECEPTOR KINASE1 (OsCERK1) to impair their chitin-induced dimerization, suppressing host immunity exerted at the lemma and palea for gaining access to the stamen and pistil. Conversely, pretreatment on spikelets with chitin induces a defense response in the lemma and palea, promoting resistance against U. virens. Collectively, our data uncover a mechanism for a U. virens virulence factor and the critical location of the host-pathogen interaction in flowers and provide a potential strategy to control rice false smut disease.

Responses of plant immune system and rhizosphere soil microbiome to the elicitor BAR11 in Arabidopsis thaliana

Microbial elicitors have been shown to boost plant immunity by inducing defense responses to reduce plant disease. However, little is known about the changes in plant microbiome and metabolism in the process of enhancing plant immunity with elicitors. The protein elicitor BAR11, from Saccharothrix yanglingensis Hhs.015, induces defense responses in Arabidopsis thaliana that enhances resistance to pathogens. In this study, bar11 was inserted into Col-0 A. thaliana to obtain BAR11-Trans plant by Agrobacterium-mediated immersion transformation. BAR11-Trans exhibited an elevated defense level against Pseudomonas syringae pv. tomato DC3000 while experiencing a decline in biomass production of above-ground parts. In the process, BAR11-Trans increased the activity of phenylalanine ammonia lyase (PAL) and catalase (CAT), and up-regulated genes related to plant defense pathways. Furthermore, BAR11-Trans decreased root tip reactive oxygen species (ROS) levels while increasing ROS burst in the leaves. Soil transplantation experiments showed that soil planted with BAR11-Trans could enhance the resistance of Col-0 A. thaliana to DC3000. Analysis of A. thaliana rhizosphere soil through 16S rRNA amplified sequencing revealed that BAR11-Trans increased the relative abundance and diversity of the rhizosphere microbial community, leading to the recruitment of more plant probiotics. Additionally, the accumulation of kaempferitrin and robinin in BAR11-Trans influenced the physicochemical properties of rhizosphere soil and the composition of the bacterial community. In summary, BAR11-Trans exhibited heightened defense levels compared to Col-0, leading to increased secretion of secondary metabolites and the recruitment of a greater number of microorganisms to adapt to the environment. These findings offer novel insights for the potential application of elicitors in agricultural disease control.

Systemic resistance induced in tomato plants by Beauveria bassiana-derived proteins against tomato yellow leaf curl virus and aphid Myzus persicae.

Tomato (Solanum lycopersicum L.) is an economically important vegetable crop around the globe. Tomato yellow leaf curling (TYLC) is the most devastating viral disease posing a serious threat to tomato production throughout the tropical and subtropical world. Induction of microbe-mediated systemic resistance in plants has been of great interest in recent years as a novel microbiological tool in disease and insect pest management. This in-vitro study aimed to determine the effectiveness of different strains (BB252, BB72 and ARSEF-2860) of a hypocreal fungus Beauveria bassiana against TYLCV disease and aphid Myzus persicae. Potted tomato plants exogenously treated with conidial and filtrate suspensions of B. bassiana strains and of their partially purified or purified proteins were exposed to TYLCV inoculum and aphid M. persicae. Results showed a significant suppression of TYLCV disease severity index by the exogenous application of conidial, filtrate and protein treatments of all B. bassiana strains and this response was directly proportional to the treatment concentration. Similarly, mean fecundity rate of M. persicae was also significantly reduced by the highest concentration of ARSEF-2860-derived elicitor protein PeBb1, followed by the highest concentrations of BB252- and BB72-derived partially purified proteins. Moreover, these B. bassiana-derived proteins also caused a significant upregulation of most of the plant immune marker genes associated with plant defense. Overall, the study findings suggest that these B. bassiana strains and their partially purified or purified elicitor proteins could be effective biological tools for the management of TYLCV and aphid infestation on tomato plants. © 2023 Society of Chemical Industry.

A protein elicitor PeVn1 from Verticillium nonalfalfae HW recognized as a MAMP triggers plant immunity response.

Protein elicitors can induce plant systemic resistance to pathogens. The recognition of a potential elicitor activates intracellular signaling events, leading to plant resistance against pathogens. In this study, a novel protein elicitor was isolated from the culture filtrate of Verticillium nonalfalfae and named PeVn1, which can induce cell death in several plant species. The PeVn1 gene was then cloned and expressed in Escherichia coli. The recombinant protein PeVn1 triggers cell death in Nicotiana benthamiana in NbBAK1 and NbSOBIR1 dependent manner. Through bioassay analysis showed that the recombinant PeVn1 induced early defense induction events, such as reactive oxygen species burst, callose deposition and the activation of defense hormone signaling pathways and defense enzyme activities. Moreover, PeVn1 significantly enhanced resistance of Nicotiana benthamiana to Sclerotinia sclerotiorum, Botrytis cinerea and N. benthamiana mosaic virus and tomato to Pseudomonas syringae pv. Tomato DC3000. In conclusion, our study reveals that PeVn1 protein as a microbe-associated molecular pattern can induce plant immune responses, which provides a theoretical basis for the development of novel protein-induced disease resistance agents.

Detection of Secreted Effector Proteins from Phelipanche aegyptiaca During Invasion of Melon Roots.

Parasites can interact with their host plants through the induction and delivery of secreted effector proteins that facilitate plant colonization by decomposing plant cell walls and inhibiting plant immune response to weaken the defense ability of the host. Yet effectors mediating parasitic plant-host interactions are poorly understood. Phelipanche aegyptiaca is an obligate root parasite plant causing severe yield and economic losses in agricultural fields worldwide. Host resistance against P. aegyptiaca occurred during the attachment period of parasitism. Comparative transcriptomics was used to assess resistant and susceptible interactions simultaneously between P. aegyptiaca and two contrasting melon cultivars. In total, 2,740 secreted proteins from P. aegyptiaca were identified here. Combined with transcriptome profiling, 209 candidate secreted effector proteins (CSEPs) were predicted, with functional annotations such as cell wall degrading enzymes, protease inhibitors, transferases, kinases, and elicitor proteins. A heterogeneous expression system in Nicotiana benthamiana was used to investigate the functions of 20 putatively effector genes among the CSEPs. Cluster 15140.0 can suppress BAX-triggered programmed cell death in N. benthamiana. These findings showed that the prediction of P. aegyptiaca effector proteins based on transcriptomic analysis and multiple bioinformatics software is effective and more accurate, providing insights into understanding the essential molecular nature of effectors and laying the foundation of revealing the parasite mechanism of P. aegyptiaca, which is helpful in understanding parasite-host plant interaction.

Protein elicitor GP1pro targets aquaporin NbPIP2;4 to activate plant immunity.

The novel protein elicitor GP1pro is the protein component of glycoprotein GP-1 isolated and identified from Streptomyces kanasensis ZX01. GP1pro induces the production of reactive oxygen species (ROS) and a hypersensitive response (HR), along with the accumulation of resistance-related genes and secondary metabolites. It ultimately regulates plant defence responses. Further analysis revealed that GP1pro interacts with the PIP2-family aquaporin protein NbPIP2;4 on the plant plasma membrane (PM) in Nicotiana benthamiana. PM localization is necessary for inducing GP1pro resistance. These results demonstrate that NbPIP2;4 acts as a H2 O2 transporter to positively regulate plant immunity and ROS accumulation. In summary, this study elucidates a conserved and novel pathway caused by GP1pro to initiate host cellular defences by targeting the plant aquaporin protein NbPIP2;4 and transporting apoplast-to-cytoplast H2 O2 to regulate plant immunity.

A novel protein elicitor (PeSy1) from Saccharothrix yanglingensis induces plant resistance and interacts with areceptor-like cytoplasmic kinase in Nicotiana benthamiana.

Previously, we reported a rare actinomycete Saccharothrix yanglingensis Hhs.015 with strong biocontrol ability, which can colonize plant tissues and induce resistance, but the key elicitor and immune mechanisms were unclear. In this study, a novel protein elicitor screened from the genome of Hhs.015, PeSy1 (protein elicitor of S.yanglingensis 1), could induce a strong hypersensitive response (HR) and resistance in plants. The PeSy1 geneencodes an 11 kDa protein with 109 amino acids that is conserved in Saccharothrix species. PeSy1-His recombinant protein induced early defence events such as acellular reactive oxygen species burst, callose deposition, and the activation of defence hormone signalling pathways, which enhanced Nicotiana benthamiana resistance to Sclerotinia sclerotiorum and Phytophthora capsici,and Solanum lycopersicum resistance to Pseudomonas syringae pv. tomato DC3000. Through pull-down and mass spectrometry, candidate proteinsthat interacted with PeSy1 were obtainedfrom N.benthamiana. We confirmed theinteraction between receptor-like cytoplasmic kinase RSy1 (Response to PeSy1) and PeSy1 using co-immunoprecipitation, bimolecular fluorescence complementation, and microscale thermophoresis. PeSy1 treatment promoted up-regulation of marker genes in pattern-triggered immunity. The cell death it elicited was dependent on the co-receptors NbBAK1 and NbSOBIR1, suggesting that PeSy1 acts as a microbe-associated molecular pattern from Hhs.015. Additionally, RSy1 positively regulated PeSy1-induced plants resistant to S.sclerotiorum. In conclusion, our results demonstrated a novel receptor-like cytoplasmic kinase in the plant perception of microbe-associated molecular patterns, and the potential of PeSy1 in induced resistance provided a new strategy for biological control of actinomycetes in agricultural diseases.

Sublethal effects of Brevibacillus laterosporus an emerging novel entomopathogenic bacterium with potential role to trigger defense response in chili pepper Capsicum annuum L. against potato aphid Macrosiphum euphorbiae

The emerging elicitor protein PeBL1, extracted from Brevibacillus laterosporus A60, was tested against the potato aphid (Macrosiphum euphorbiae) for its biocontrol potential. Its effects on M. euphorbiae survival, lifespan, immature development, adult reproductive performance, and pest–pathogen interaction were assessed using electrical penetration graphs (EPGs), The levels of jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) were significantly increased in the B. laterosporus-treated C. annuum seedlings, indicating substantial accumulation. Brevibacillus laterosporus improved the height of the C. annuum L. plant and the surface structure of the leaves, by reducing M. euphorbiae reproduction and preventing colonization, according to the findings of this study. The activation of pathways was also part of the defensive response (JA, SA, and ET). The use of B. laterosporus for integrated pest management in the agroecosystem appears to be suitable based on these findings against M. euphorbiae.

PeVL1 Novel Elicitor Protein, from Verticillium lecanii 2, Enhances Systemic Resistance against Rice Leaf Roller (Marasmia ruralis Wlk.) in Rice (Oryza sativa L.).

The hazardous pest known as rice leaf roller (Marasmia ruralis Wlk.) (Lepidoptera: Pyralidae), which undermines rice (Oryza sativa L.) output globally, folds the leaves of the rice plant. Protein elicitors are thought to be biological elements that causes the rice to become resistant to herbivores. The potential for biocontrol of the emerging elicitor protein evaluated from Verticillium lecanii 2 (PeVL1) was evaluated against M. ruralis. To assess the impact of PeVL1 on immature development, survival, and lifetime, four different PeVL1 concentrations were allocated. Electrical penetration graphs (EPGs) against M. ruralis were used to evaluate adult reproductive efficiency and the interaction between the pest and the pathogen. Furthermore, the characterization of active substances in PeVL1 with multi-acting entomopathogenic effects looked into the direct interactions of PeVL1 with temperature and climatic change in rice (O. sativa) plants. PeVL1 treatments reduced the population increase of second and third generation M. ruralis compared to controls. In a test of host selection, M. ruralis colonized control plants more quickly than PeVL1-treated O. sativa plants. PeVL1 concentrations prolonged the M. ruralis larval stage. Similar to fecundity, PeVL1-treated seedlings produced fewer offspring than control seedlings. On PeVL1-treated leaves, trichomes and wax production created an unfavorable habitat for M. ruralis. PeVL1 changed the surface structure of the leaves, which inhibited colonization and decreased M. ruralis reproduction. The activation of pathways was another aspect of systemic defense activities including jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). Based on these results against M. ruralis, the use of PeVL1 in the agroecosystem with integrated pest management and biocontrol seems appropriate. Our research provides a novel insight into a cutting-edge biocontrol method utilizing V. lecanii 2.

Whole-Genome Resource of Lasiodiplodia pseudotheobromae BaA, the Causative Agent of Black Root Rot Morinda officinalis.

Whole-Genome Resource of Lasiodiplodia pseudotheobromae BaA, the Causative Agent of Black Root Rot Morinda officinalis.

Chickpea pathogen-resistance-rating correlates with responsiveness to an immune elicitor protein from Phytophthora medicaginis

Identifying elicitors of plant defence from soilborne plant pathogens can provide valuable knowledge about plant-microbe interactions and can be used as an efficient screening tool in breeding programs to identify pathogen resistant genotypes. Phytophthora medicaginis causes Phytophthora Root Rot (PRR) in a range of Fabaceae hosts including soybean and chickpea. The P. medicaginis:soybean pathosystem has been well studied and qualitative plant resistance loci identified, however less is known concerning the interaction with chickpea and only quantitative resistance has been identified to date. We hypothesized that P. medicaginis encodes elicitors that are perceived by chickpea and that the responsiveness of different genotypes to the elicitor(s) would vary with disease resistance. Using sequential purification, we successfully isolated proteinaceous fractions that contained elicitor molecule(s) from the mycelium of Phytophthora medicaginis but not from the culture supernatant. We were able to purify a ∼22 kDa protein from these fractions that induced three classical plant markers for elicitor perception: an average of 2.5x more ethylene than control treatment, deposition of callose, and an increase of 2.1x in phenylalanine ammonia-lyase activity. PmEip1 perception was found to elicit strong responses in other Fabaceae hosts, and that its activity in chickpea plants varied with the degree of PRR susceptibility whereby chickpea varieties with higher levels of PRR resistance were more responsive to PmEip1 than susceptible varieties. Using tandem mass spectrometry, we found that PmEip1 shared 87.6% homology to a glyceraldehyde-3-phosphate dehydrogenase from Phytophthora cactorum. Therefore, P. medicaginis produces an elicitor protein that is perceived by chickpea. This elicitor protein could be used as a means of screening for resistant genotypes in future chickpea breeding programs.

Differential plant response to toxins and elicitor proteins released by the potato and tomato pathogens Alternaria solani and Alternaria alternata

Early blight of potato and tomato, caused principally by Alternaria solani, results in extensive damage to foliar tissues. Symptoms are seen as expanding necrotic lesions. Lesions are sometimes surrounded by a halo of chlorotic tissue, however the basis for presence or absence of the distinctive halo have not been determined. To dissect the basis for lesion development a series of Alternaria-associated toxins and potential elicitor proteins were tested in potato and tobacco. Infiltration of leaves demonstrated that, while most of the pure toxins did not have a visible effect, two anthraquinones, bostrycin and altersolanon A, elicited a strong necrosis-mediated response. Extracts of solid and liquid cultures of A. solani yielded anthraquinones that also elicited a necrotic response. Two potential elicitor proteins were identified and cloned from the potato brown spot and early blight pathogens Alternaria alternata and A. solani, respectively. Agrobacterium-mediated infiltration and expression of the A. alternata xyloglucanase cel12-A and A. solani Hrip1 revealed that cel12-A had no apparent effect, whereas Hrip1 induced expanding tissue death and development of chlorotic halos. Development of chlorotic halos was dependent on leaf age, with older leaves exhibiting halos. It is proposed that specific toxin and elicitor production, in conjunction with tissue susceptibility mediate the outcomes of early blight symptoms.

Biocontrol Potential of PeBL2, a Novel Entomopathogenic Bacterium from Brevibacillus laterosporus A60, Induces Systemic Resistance against Rice Leaf Folder Cnaphalocrocis exigua (Butler) in Rice (Oryza sativa L.)

The dangerous insect pest known as rice leaf folder Cnaphalocrocis exigua (Butler), which reduces rice output globally, twists and feeds on the young rice plant's leaves. Protein elicitors are hypothesized to be biological components that promote rice in becoming herbivore resistant. The evolving elicitor protein PeBL2, obtained from Brevibacillus laterosporus A60, was tested for biocontrol against C. exigua. Four distinct PeBL2 doses (74.23, 45.53, 22.26, and 11.13 μg mL-1) were assigned to evaluate the impact of PeBL2 on immature growth, survivability, and lifespan. Adult reproductive efficiency and the interaction between the pest and the disease were assessed against C. exigua. Further, the assessment of active compounds in PeBL2 with multi-acting entomopathogenic effects investigated the direct correlations of PeBL2 with temperature and climatic change in plants of rice (Oryza sativa L.). When compared to controls, PeBL2 treatments reduced the growing population of second- and third-generation C. exigua. Cnaphalocrocis exigua colonized control plants faster than PeBL2-treated O. sativa plants in a host selection test. PeBL2 doses delayed the development of the larval stage of C. exigua. PeBL2-treated seedlings generated less offspring than control seedlings, identical to fecundity. Trichomes and wax formation on PeBL2-treated leaves generated an adverse environment for C. exigua. PeBL2 altered the surface topography of the leaves, preventing colonization and reducing C. exigua reproduction. PeBL2-treated O. sativa seedlings exhibited somewhat increased amounts of jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). Systemic defensive processes also included the activation of pathways (JA, SA, and ET). Following these results versus C. exigua, the use of PeBL2 in an agroecosystem with integrated pest management and biocontrol appears to be reasonable. These findings shed new light on a cutting-edge biocontrol technique based on B. laterosporus A60.

A novel elicitor protein phosphopentomutase from Bacillus velezensis LJ02 enhances tomato resistance to Botrytis cinerea.

The loss of tomatoes caused by Botrytis cinerea (B. cinerea) is one of the crucial issues restricting the tomato yield. This study screened the elicitor protein phosphopentomutase from Bacillus velezensis LJ02 (BvEP) which improves the tomato resistance to B. cinerea. Phosphatemutase was reported to play a crucial role in the nucleoside synthesis of various microorganisms. However, there is no report on improving plant resistance by phosphopentomutase, and the related signaling pathway in the immune response has not been elucidated. High purity recombinant BvEP protein have no direct inhibitory effect on B. cinerea in vitro,and but induce the hypersensitivity response (HR) in Nicotiana tabacum. Tomato leaves overexpressing BvEP were found to be significantly more resistant to B. cinerea by Agrobacterium-mediated genetic transformation. Several defense genes, including WRKY28 and PTI5 of PAMP-triggered immunity (PTI), UDP and UDP1 of effector-triggered immunity (ETI), Hin1 and HSR203J of HR, PR1a of systemic acquired resistance (SAR) and the SAR related gene NPR1 were all up-regulated in transgenic tomato leaves overexpressing BvEP. In addition, it was found that transient overexpression of BvEP reduced the rotting rate and lesion diameter of tomato fruits caused by B. cinerea, and increased the expression of PTI, ETI, SAR-related genes, ROS content, SOD and POD activities in tomato fruits, while there was no significant effect on the weight loss and TSS, TA and Vc contents of tomato fruits. This study provides new insights into innovative breeding of tomato disease resistance and has great significance for loss reduction and income enhancement in the tomato industry.

A novel elicitor MoVcpo is necessary for the virulence of Magnaporthe oryzae and triggers rice defense responses.

Rice blast caused by Magnaporthe oryzae is one of the most important diseases of rice. Elicitors secreted by M. oryzae play important roles in the interaction with rice to facilitate fungal infection and disease development. In recent years, several elicitor proteins have been identified in M. oryzae, and their functions and importance are increasingly appreciated. In this study, we purified a novel elicitor-activity protein from M. oryzae, which was further identified as a vanadium chloroperoxidase (MoVcpo) by MAIDL TOF/TOF MS. The purified MoVcpo induced reactive oxygen species (ROS) accumulation in host cells, up-regulated the expression of multiple defense-related genes, thus significantly enhancing rice resistance against M. oryzae. These results suggested that MoVcpo functions as a pathogen-associated molecular pattern (PAMP) to trigger rice immunity. Furthermore, MoVcpo was highly expressed in the early stage of M. oryzae infection. Deletion of MoVcpo affected spore formation, conidia germination, cell wall integrity, and sensitivity to osmotic stress, but not fungal growth. Interestingly, compared with the wild-type, inoculation with MoVcpo deletion mutant on rice led to markedly induced ROS accumulation, increased expression of defense-related genes, but also lower disease severity, suggesting that MoVcpo acts as both an elicitor activating plant immune responses and a virulence factor facilitating fungal infection. These findings reveal a novel role for vanadium chloroperoxidase in fungal pathogenesis and deepen our understanding of M. oryzae-rice interactions.

Prokaryotic expression, purification and activity analysis of necrosis and ethylene-inducing peptide 1 (Nep1)-like protein from <italic>Magnaporthe grisea</italic>

<p indent="0mm">Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are kinds of secreted by a range of plant-associated microorganisms including bacteria, fungi, and oomycetes. The first member of NLP families is Nep1 protein isolated and purified from the culture filtrates of <italic>Fusarium oxysporum</italic> by Bailey in 1995. The protein induced ethylene and necrosis in leaves of a variety of dicotyledoneae but not in members of the monocotyledoneae tested. In addition, the Nep1 and some members of NLP family can also induce defense-related responses, including the expression of pathogenesis-related genes, callose deposition, accumulation of reactive oxygen species (ROS), and hypersensitive-like response in plants. Previous studies have shown that the Nep1 and some members of NLP family play dual roles in plant-pathogen interactions. They not only act as a cytolytic toxin that causes plasma membrane permeabilization and cytolysis in plant cells but also represent a class of microbial-associated molecular patterns (MAMPs) that trigger plant innate immune response. Many plant pathogen genomes contain different numbers and types of NLP family members. Different pathogens differ significantly in the number of NLP members, for example, the genome of <italic>Mycosphaerella graminicola</italic> contains only a single NLP homologue, whereas the number of NLP members in the genome of<italic> Phytophthora sojae</italic> and <italic>Phytophthora capsici</italic> up to 33 and 39, respectively. Recently, the NLP toxins are reported to target the plant cell-surface GIPC sphingolipids, and the difference in predominant GIPC series produced by the Eudicot and monocot determine host selectivity of microbial NLP cytolysins. However, recently, some NLP toxins produced by the pathogen of monocot were also proved to have cytotoxic activity on various monocot plant species and legitimize their presence in monocot-specific plant pathogens. The<italic> Magnaporthe grisea</italic>, causal agent of rice blast disease, contains four NPP1 domain-containing gene (MoNLP) in the fully sequenced genome. Although they are all highly induced during the infection of rice, the quadruple ΔMoNLP <italic>M</italic>.<italic>griseae</italic> mutant strains do not compromise its virulence. Therefore, the function and significance of their presence are still largely unknown. To further explore the potential function of these NLP members during the interactions between rice and <italic>Magnaporthe oryzae</italic> as well as the possibility that they may act as potential plant immunity inducers to modulate the plant immune response, the coding region sequences were cloned and ligated into the pGEX-6P-1 vector to prokaryotically express and purify these proteins by analyzing their sequence through bioinformatics methods. The results indicated that the solubility of prokaryotically expressed protein decreased obviously with the induction temperature increasing, but the expressed recombinant protein also increased. These corresponding purified and soluble proteins were then further infiltrated into the leaves of tobacco, indicated that the MoNLP1 and MoNLP4 fused with N-terminal GST tag were bioactive and induced the obvious leaf tissue necrosis of tobacco. However, the purified GST-MoNLP2 protein did not induce tissue necrosis in tobacco leaves, which may be due to expressed fusion protein with unexcised signal peptide or low protein concentration for injection. Our study laid a foundation for further investigating the function of this gene family in rice blast fungus and exploiting more potential protein elicitors for plants.