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Xenorhabdus budapestensis strain C72 isolated from the entomopathogenic nematode of Steinernema bicornutum possesses an excellent biocontrol effect on southern corn leaf blight. However, its genomic information is lacking. Here, we report a high-quality complete and annotated genome sequence of X. budapestensis strain C72. Fifteen secondary metabolite biosynthetic gene clusters are identified in the genome, which are responsible for the production of a diverse group of antimicrobial compounds to help host plants against agricultural pathogenic diseases. This genome sequence could contribute to investigations of the molecular basis underlying the biocontrol activity of this Xenorhabdus strain.

Bacterial microbe-associated molecular patterns (MAMPs) play an important role in innate plant immunity. This in vitro study evaluated the putative role of protein elicitor PeBA1 derived from Bacillus amyloliquefaciens NC6 strain in eliciting induced resistance type responses in common bean (Phaseolus vulgaris) plants against green peach aphid Myzus persicae. Nymphal developmental time of aphids was significantly prolonged and the fecundity was significantly reduced by different concentrations of PeBA1 elicitor (i.e. 40.51, 24.91 and 16.38 µg mL-1) applied at three different temperature regimes (i.e. 21, 27 and 30 °C). Moreover, foliar application of PeBA1 elicitor protein strongly up-regulated the expression levels of salicylic acid (SA) pathway-associated genes, while the expression levels of jasmonic acid (JA) pathway-associated genes exhibited a moderate induction. Quantification by LC/MSMS revealed a linear increase of both SA and JA plant defense hormones along with the time of exposure. Our findings suggest that the bacterial elicitor protein PeBA1 could be used as an effective biological pest management tool against phloem-feeding insect pests such as green peach aphids M. persicae.

Drought stress, as a key negative factor, could result in the reduction of yield and quality in rice. Myo-inositol oxygenase (MIOX), a unique monooxygenase, exerts an essential role in plant drought resistance. To investigate the function of the OsMIOX gene in drought process, a transcriptomic profile between OsMIOX-overexpressing (OE) plants and wild-type (WT) was performed by RNA-sequencing in rice. Under drought stress, a total of 23 million high-quality reads, assembled into 28,552 unigenes, were generated and 743 genes (534 up-regulated and 209 down-regulated) were significantly differentially expressed between OE plants and WT. These detected genes were mainly associated with plant hormones transduction and sugar metabolism. Interestingly, several transcription factors (TFs) were prominently identified in OE lines. A significant positive correlation between RNA-Seq data and qRT-PCR results was identified. In all, our results could partially provide relatively valuable clues in identifying candidate genes involved in drought-stress resistance and elucidating basis theory underlying the role of OsMIOX in drought resistance.

Whiteflies, Bemisia tabaci Gennadius (Aleyrodidae: Hemiptera), are a polyphagous economically destructive pest of several solanaceous crops around the world. Many secondary metabolites are synthesized by different biotrophic and necrotrophic fungi which are capable of inducing systemic resistance in plants against various phytophagous pests. The present laboratory work demonstrated the anti-insect impact of a protein extracted and purified partially from an entomopathogenic fungus (EPF) Lecanicillium lecanii (Zimmermann) against B. tabaci. Three different concentrations (i.e., 7.43, 11.15, and 22.31 μg mL−1) of this protein were bioassayed to assess its effect on the fecundity rate of B. tabaci on cotton (Gossypium hirsutum L.) plants. Furthermore, the possible implication of this fungal protein in defense pathways of cotton plants was evaluated by determining the expression profiles of salicylic acid (SA) and jasmonic acid (JA) pathways related to major genes through reverse transcription qPCR (RT-qPCR). According to the results, all protein concentrations exerted a significant (F3, 252 = 62.51, p ≤ 0.001) and negative impact on the fecundity rate of B. tabaci females. At the highest protein concentration (22.31 μg mL−1), the minimum rate of fecundity (i.e., 2.46 eggs female−1day−1) of B. tabaci was noted on the seventh day, whereas fecundity rates for the other two protein concentrations (i.e., 11.15 and 7.43 μg mL−1) were, respectively, 3.06 and 3.90 eggs day−1 female−1. The maximum rate of fecundity (6.01 eggs female−1day−1) was recorded in untreated (control) treatments. In addition, the foliar application of L. lecanii derived protein significantly upregulated all SA linked genes (OPR3, PPO1 and COI1) and slightly triggered up the JA linked genes (LOX1, UBQ7 and AOS) in the cotton plants. These findings revealed that this L. lecanii extracted partially purified protein triggered systemic resistance against B. tabaci in the cotton plants, proposing its putative effectiveness as an innovative biological control technique against B. tabaci and other phloem-feeding hemipteran pests. Nevertheless, further investigations such as purification and molecular and functional characterization of this L. lecanii-derived partially purified protein are required.

MoHrip2, identified from Magnaporthe oryzae as an elicitor, can activate plant defense responses either in the form of recombinant protein in vitro or ectopic expressed protein in rice. However, its intrinsic function in the infective interaction of M. oryzae-rice is largely unknown. Here, we found that mohrip2 expression was significantly induced at stages of fungal penetration and colonization. Meanwhile, the induced MoHrip2 mainly accumulated in the rice apoplast by outlining the entire invasive hyphae during infection, and its secretion was via the conventional endoplasmic reticulum (ER)-to-Golgi pathway, demonstrating the nature of MoHrip2 as an apoplastic effector. What's more, the disease facilitating function of MoHrip2 was revealed by the significantly compromised virulence of Δmohrip2 mutants on rice seedlings and even on the wounded rice leaves. Inoculations of these mutant strains on rice leaf sheaths showed a reduction in penetration and subsequent expansion of fungal growth, which is probably due to activated host immunity including the expression of certain defense-related genes and the production of certain phytoalexins. Altogether, these results demonstrated the necessity of MoHrip2 in suppression of host immunity and the full virulence of M. oryzae.

Plants rely on various physiological and molecular defense mechanisms against biotic stresses such as herbivore insects. Many entomopathogenic fungi synthesize protein molecules that can trigger these plant defenses. This laboratory study characterized the bioactivity of a partially purified protein derived from Beauveria bassiana (ARSEF 2860) against whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), which is an economically important pest of agricultural and horticultural crops worldwide. Different concentrations (i.e., 0.021, 0.042 and 0.063 &mu, M) of fungal protein were bioassayed to determine their sub-lethal effect on the survival percentage and fecundity rate of B. tabaci on tomato (Lycopersicon esculentum) plants. In addition, the putative role of this partially purified B. bassiana protein in the defense mechanisms of plant was assessed through the expression analyses of important genes related to salicylic acid (SA)&mdash, and jasmonic acid (JA)&mdash, associated pathways using RT-qPCR. Results revealed a significant suppression of the survival percentage and fecundity rate of B. tabaci by the fungal protein. Lowest survival (41%) was recorded for the highest concentration of protein (0.063 &mu, M), whereas mean survival for the other two protein concentrations (0.042 and 0.021 &mu, M) were 62 and 71%, respectively. Likewise, the highest and lowest mean fecundity rates were observed for the control and the highest protein concentration (i.e., 3.3 and 1.8 eggs day&minus, 1 female&minus, 1, respectively). Furthermore, the exogenous application of B. bassiana-derived protein on tomato plants strongly up-regulated the SA-related genes (PAL, PR1, BGL2 and EDS1) and slightly up-regulated the JA-related genes (AOC, AOS, OPR3 and LOX) as compared to the control plants. These findings demonstrate the putative role of this partially purified B. bassiana protein fraction in inducing systemic resistance in the tomato plants against B. tabaci, suggesting its further purification and characterization to be used as novel biological pest control tool against B. tabaci and other sap-sucking insect pests.

A multipartite DNA virus was isolated from a plant pathogenic fungus, which is infectious as cloned DNA and purified virions., Here, we describe a tripartite circular single-stranded (ss) DNA mycovirus, named Fusarium graminearum gemytripvirus 1 (FgGMTV1). The genome of FgGMTV1 comprises three circular ssDNA segments (DNA-A, DNA-B, and DNA-C). Sequence alignments and phylogenetic analyses showed that FgGMTV1 is nested within the family Genomoviridae. We also constructed the first infectious DNA clones of a DNA mycovirus. Our results show that DNA-A and DNA-B are mutually interdependent for their replication and are associated with severely reduced colony growth and hypovirulence. DNA-C relies on DNA-A and DNA-B for replication and is necessary for the recovery of abnormal fungal phenotypes. DNA-C also enhances the accumulation of viral DNA in infected fungi and permits stable colonization and easy transmission via conidia. This is the first multipartite DNA virus isolated from a fungus. Our phylogenetic analyses also suggest that the multipartite genome of FgGMTV1 may have evolved from a monopartite genome of an ancient genomovirus.

Myzus persicae, a destructive aphid of tomato usually managed by chemical pesticides, is responsible for huge annual losses in agriculture. In the current work, a protein elicitor, PeBL1, was investigated for its capacity to induce a defense response against M. persicae in tomato. Population growth rates of M. persicae (second and third generation) decreased with PeBL1 treatments as compared with controls. In a host selection assay, M. persicae showed preference for colonizing control plants as compared to tomato seedlings treated with PeBL1. Tomato leaves treated with PeBL1 gave rise to a hazardous surface environment for M. persicae due to formation of trichomes and wax. Jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) showed significant accumulation in tomato seedlings treated by PeBL1. The following results showed that PeBL1 significantly modified the tomato leaf surface structure to reduce reproduction and deter colonization by M. persicae. Defense processes also included activation of JA, SA, and ET pathways. The study provides evidence for use of PeBL1 in the protection of tomato from M. persicae.

PevD1, an Aa1-like protein secreted by Verticillium dahlia, promotes infection by inhibiting the antifungal activity of Gossypium hirsutum PR5. ., Alt a 1 family proteins (AA1s) have only been observed in the Dothideomycetes and Sordariomycetes classes of fungi, and their biological functions have remained poorly understood. Verticillium dahliae, a soil-borne pathogen that causes plant wilt disease, secretes hundreds of proteins during the process of pathogenic infection, including the AA1 member PevD1. In this study, we found that the pevd1 transcript was present in all of the hosts studied (cotton, Arabidopsis, tomato, and tobacco) and showed elevated expression throughout the infection process. Furthermore, pevd1 knockout mutants displayed attenuated pathogenicity compared with the wild-type (WT) strain and complemented strains in hosts. A partner protein of PevD1, pathogenesis-related protein 5 (PR5)-like protein GhPR5, was isolated from cotton (Gossypium hirsutum) plants by co-purification assays, and the PevD1–GhPR5 interaction was determined to be localized in the C-terminus (PevD1b, amino acids residues 113–155) by pull-down and yeast two-hybrid techniques. Re-introduction of the pevd1b gene into a pevd1 knockout mutant resulted in restoration of the virulence phenotype to WT levels. In addition, PevD1b, which is similar to PevD1, decreased the antifungal activity of GhPR5 in vitro. Our findings reveal an infection strategy in which V. dahliae secretes PevD1 to inhibit GhPR5 antifungal activity in order to overcome the host defence system.

The Botrytis cinerea BcSpl1 protein is a member of the cerato-platanin family, and consists of 137 amino acids and two disulfide bridges. This protein induces the onset of necrosis in infiltrated plant hosts. Recombinant BcSpl1 proteins produced in Pichia pastoris (pBcSpl1) and Escherichia coli (eBcSpl1) were initially compared regarding their abilities to induce necrosis and systemic acquired resistance (SAR). The pBcSpl1 and eBcSpl1 treatments led to the development of necrotic lesions on tomato leaves, and provided tomato plants with SAR to B. cinerea. The lesion area of leaves infiltrated with the BcSpl1 proteins decreased by 22.7% (pBcSpl1) and 21.8% (eBcSpl1). Additionally, eBcSpl1 up-regulated the expression levels of some defense-related genes, including PR-1a, prosystemin, PI I, and PI II, as well as SIPK and TPK1b, which encode two protein kinases. Furthermore, eBcSpl1 exhibited chitin-binding properties. Our data revealed that the E. coli expression system produces higher BcSpl1 yields than the P. pastoris system. This high-yield expression of BcSpl1 may be relevant for future large-scale applications of this elicitor to improve crop production.

PevD1, a fungal effector secreted by Verticillium dahliae, could induce hypersensitive responses-like necrosis and systemic acquired resistance (SAR) in cotton and tobacco plants. PevD1 could drastically induce the expression of Nbnrp1, which is an asparagine-rich protein (NRP) of Nicotiana benthamiana. Our previous research indicated that Nbnrp1 positively regulated PevD1-induced cell necrosis and disease resistance. In this study, we further investigated PevD1-induced immune responses in both wild-type (WT) and Nbnrp1-RNAi lines through RNA-seq, in order to reveal the underlying mechanism of Nbnrp1-modulated PevD1-induced disease resistance in N. benthamiana. Results showed that Nbnrp1-RNAi lines exhibited reduced PevD1-induced immune responses, like inhibiting H2O2 accumulation and MAPK phosphorylation. To silence Nbnrp1 inhibited the expression of PevD1-induced differential expression genes (DEGs) involved in pathways associated with sesquiterpenoid and triterpenoid biosynthesis, flavone and flavonol biosynthesis, plant-pathogen interaction and phenylpropanoid biosynthesis, etc. It is worth noting that sesquiterpene phytoalexin capsidiol accumulation were obviously decreased in Nbnrp1-RNAi plants after PevD1 treatment, accompanied with the down-expression of EAS and EAH, which were two key genes related to capsidiol biosynthesis. These results suggested that Nbnrp1 mediates PevD1-induced defense responses by regulating sesquiterpenoid phytoalexins biosynthesis pathway.

Phytophthora species cause enormous economic loss every year worldwide. Xenocoumacin 1 (Xcn1), isolated from the bacterium Xenorhabdus nematophilus, is a broad-spectrum antibiotic against agricultural pathogens, especially Phytophthora. To understand the inhibitory mode of Xcn1 toward Phytophthora pathogens, we determined the inhibitory effects of Xcn1 on Phytophthora capsici both in vitro and in vivo. In vitro, Xcn1 inhibited different stages in the life cycle of P. capsici, including sporangium formation, zoospore germination, and mycelial growth, with 50% effective concentration (EC50) values of 0.037, 0.81, and 2.44 μg ml−1, respectively. Xcn1 also reduced zoospore motility. In vivo, Xcn1 efficiently controlled the Phytophthora blight of pepper with a disease reduction of 99% at a concentration of 5 μg ml−1 assessed on the third day after incubation of wound stem plants. In addition, Xcn1-treated P. capsici mycelia exhibited increased mycelial branch spacing, evident plasmolysis, and leakage of intracellular components. In conclusion, in the presence of Xcn1, several stages in the life cycle of P. capsici were inhibited, and the hyphae exhibited obvious morphological changes.

The protein elicitor PevD1, isolated from Verticillium dahlia, could enhance resistance to TMV in tobacco and Verticillium wilt in cotton. Here, the pevd1 gene was over-expressed in wild type (WT) Arabidopsis, and its biological functions were investigated. Our results showed that the transgenic lines were more resistant to Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 than the WT line was. In transgenic plants, both the germination time and bolting time required were significantly shorter and fresh weights and plant heights were significantly higher than those in the WT line. A transcriptomics study using digital gene expression profiling (DGE) was performed in transgenic and WT Arabidopsis. One hundred and thirty-six differentially expressed genes were identified. In transgenic Arabidopsis, three critical regulators of JA biosynthesis were up-regulated and JA levels were slightly increased. Three important repressors of the ABA-responsive pathway were up-regulated, indicating that ABA signal transduction may be suppressed. One CML and two WRKY TFs involved in Ca(2+)-responsive pathways were up-regulated, indicating that this pathway may have been triggered. In conclusion, we show that PevD1 is involved in regulating several plant endogenous signal transduction pathways and regulatory networks to enhance resistance and promote growth and development in Arabidopsis.

This research reported the in vitro pathogenicity of Verticillium lecanii strains, L2 and L5, and Beauveria bassiana strains, B76 and B252, against Megoura japonica using leaf-dip method. The virulence potential of these four entomopathogenic fungal strains of V. lecanii and of B. bassiana were compared between fungi conidia (concentrations 1 &times, 106, 1 &times, 107, and 1 &times, 108 conidia mL&minus, 1) and culture filtrate. Moreover, binary combination of four different fungal strains (L2 + B76, B76 + L5, L2 + B252, and L2 + B76 + B252 + L5) were evaluated against M. japonica under control condition. Aphid mortality was recorded after two, four, six, and eight days of post-treatment. In the conidial concentration bioassay, strain B76 showed maximal mortality (85.3%) against bean aphid, and strain L5 showed the lowest effect (60.0%) at the highest concentration (1 &times, 1) at eight days post-treatment. Whereas, binary combinations of B76 and L2 strains showed the highest effect against M. japonica (90.5%) than other combinations. Moreover, in comparison with the effect of filtrate and conidia bioassay, 91.4% and 84.1% were achieved in strain B76, and the lowest effect (63.8% and 55.1%) was recorded in strain L5.

This study reports the characterization of protein elicitor PeBb1 derived from entomopathogenic fungus Beauveria bassiana ARSEF-2860 strain and its putative role in induced systemic resistance in Brassica rapa ssp. pekinensis against green peach aphid Myzus persicae. The sequence of purified elicitor protein was matched with the genomic sequence of a hypothetical protein BBA_10269 from B. bassiana ARSEF-2860 (GenBank Accession No. XP_008603588.1). The protein-encoding gene PeBb1 contained 534 bp cDNA encoding a polypeptide of 177 amino acids with a molecular mass of 19 kDa. The recombinant elicitor protein was expressed in Escherichia coli using pET-28a (+) expression vector and induced necrosis in the leaves of tobacco. The effects of elicitor protein on aphid M. persicae was determined by applying three different concentrations of PeBb1 (i.e., 26, 35, 53 &mu, M) on B. rapa plants at 4-leaf stage and the treated plants were exposed to newly emerged (0&ndash, 6 h old) apterous adult aphids. Bioassay results showed significant (p &lt, 0.05) sub-lethal effects of the exogenous application of PeBb1 elicitor on M. persicae. Moreover, the RT-qPCR gene expression analyses showed a significant up-regulation of most of the key genes linked to ethylene (ET)- and jasmonic acid (JA)-associated plant defense pathways in elicitor-treated plants. These results not only recommend the putative utilization of PeBb1 elicitor protein in future biological pest control strategies against phloem-feeding insect pests such as M. persicae, but also help in better comprehension of the mechanisms through which beneficial fungi trigger the induced plant resistance.

Many biotrophic and necrotrophic fungi synthesize proteins that may elicit induced plant resistance against different herbivore pests. This in-vitro study elucidates the sub-lethal effect of a partially-purified protein derived from the entomopathogenic fungus Lecanicillium lecanii (Zimmerman) (Hypocreales: Clavicipitaceae) against green peach aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae), an economically important pest of many solanaceous crops including tomato. Bioassays were conducted to determine the impact of different concentrations of protein (i.e. 0.018, 0.036 and 0.054 µM) on the survival and fecundity of M. persicae on tomato (Lycopersicon esculentum) plants. Moreover, the potential role of this exogenous protein in the plant defense mechanism was assessed by expression analyses of key genes associated with salicylic acid (SA) and jasmonic acid (JA) pathways using RT-qPCR. The results indicated a significant negative effect of all protein concentrations on the survivorship and fecundity of M. persicae. The highest concentration (0.054 µM) resulted in lowest survival (46%) of aphids at 7th day post-treatment, while two other concentrations (0.036 and 0.018 µM) resulted in 61 and 71% survival rate, respectively. Similarly, lowest and highest mean fecundity rates were recorded for the highest protein concentration and the control (1.5 and 2.4 nymphs day−1 female−1), respectively. Moreover, L. lecanii-derived protein strongly upregulated the SA associated genes PR1, BGL2 and PAL, and moderately upregulated the JA associated genes LOX, AOS and AOC in protein-treated tomato plants compared to the control plants. These findings demonstrate the systemic resistance induced in tomato plants against M. persicae by the exogenous application of partially-purified protein extracted from L. lecanii, suggesting its further purification and characterization as a novel biological pest management tool against aphids and other phloem-feeding insect pests.

Elicitors are biofactors that induce resistance in plants against different insect pests. This in vitro study evaluated the impact of a novel elicitor protein PeBC1, extracted from a necrotrophic fungus Botrytis cinerea, on the development and fecundity parameters of green peach aphid (Myzus persicae) on common beans (Phaseolus vulgaris L.). Three different concentrations of PeBC1 elicitor (i.e., 33.56, 25.43, 19.33 µg mL−1) were applied at three different temperature regimes (i.e., 18, 21, and 25 °C). Elicitor treatments were applied topically on the bean plants at 3-leaf stage and newly emerged (0–6 h old) apterous adult aphids were exposed to these treated leaves. In addition to the biological parameters of aphids, the relative expression levels of key genes associated with jasmonic acid (JA) and salicylic acid (SA) plant defense pathways were also determined through RT-qPCR. Results of bioassays revealed that the application of PeBC1 elicitor protein exhibited pronounced and significant (p < 0.05) sub-lethal effects on green peach aphids. The fecundity was reduced and the nymphal development time was prolonged by different concentrations of PeBC1 elicitor and temperature regimes. Gene expression studies showed that the exogenous application of PeBC1 induced a significant upregulation of the expression levels of JA and SA pathway-associated genes in bean plants. As compared to control, elicitor-treated plants exhibited an induced resistance against aphids. Our findings suggest the potential use of PeBC1 elicitor protein in future bio-intensive management strategies against sap-sucking insect pests such as green peach aphids.

Green peach aphid, Myzus persicae (Hemiptera: Aphididae), is an economically important pest of crops within more than 40 plant families all over the world. This study encompasses in-vitro pathogenicity of two strains of Beauveria bassiana (BB-72 and BB-252) and one strain of Lecanicillium lecanii (V-4) against green peach aphid (M. persicae). Using a leaf-dip method, three different bioassays were conducted comprised of filtrates and conidial concentrations of BB-72, BB-252 and V-4 fungal strains and their binary combinations. Infiltrate bioassays, 2 mL fungal filtrate of each strain was used. In conidial bioassays, three different concentrations (i.e., 1 &times, 106, 1 &times, 107 and 1 &times, 108 conidia mL&minus, 1) of each fungal strain were used, while in binary combination bioassays, LC50 and LC33 of these fungal strains were evaluated. According to the results, maximum pathogenicity against test aphids was observed at 10th day of all treatments. Both strains of B. bassiana (BB-72 and BB-252) exhibited higher mortality of aphids (up to 95 and 91%, respectively) than L. lecanii (V-4) strain (87%) in all three types of bioassays. Moreover, binary combinations of BB-72 and BB-252 strains showed significantly higher aphid mortality (94%) than other combinations. Reduced mortality in case of fungal combinations of V-4 strain might be due to the antagonistic effect of L. lecanii strain along with both strains of B. bassiana. Nevertheless, evaluation of combined pathogenicity of the fungal strains constitutes the novelty of this in-vitro study which revealed that both strains of B. bassiana (i.e., BB-72 and BB-252) are mutually compatible and can be utilized together as new biocontrol tools against destructive insect pests such as M. persicae.

Botrytis cinerea is one of the most notorious pathogenic species that causes serious plant diseases and substantial losses in agriculture throughout the world. We identified BcXyl1 from B. cinerea that exhibited xylanase activity. Expression of the BcXyl1 gene was strongly induced in B. cinerea infecting Nicotiana benthamiana and tomato plants, and BcXyl1 deletion strains severely compromised the virulence of B. cinerea. BcXyl1 induced strong cell death in several plants, and cell death activity of BcXyl1 was independent of its xylanase activity. Purified BcXyl1 triggered typically PAMP-triggered immunity (PTI) responses and conferred resistance to B. cinerea and TMV in tobacco and tomato plants. A 26-amino acid peptide of BcXyl1 was sufficient for elicitor function. Furthermore, the BcXyl1 death-inducing signal was mediated by the plant LRR receptor-like kinases (RLKs) BAK1 and SOBIR1. Our data suggested that BcXyl1 contributed to B. cinerea virulence and induced plant defense responses.

Verticillium dahliae is a wide-host-range fungal pathogen that causes soil-borne disease in hundreds of dicotyledonous hosts. In search of V. dahliae Vd991 cell death-inducing proteins, we identified a pectate lyase (VdPEL1) that exhibited pectin hydrolytic activity, which could induce strong cell death in several plants. Purified VdPEL1 triggered defense responses and conferred resistance to Botrytis cinerea and V. dahliae in tobacco and cotton plants. Our results demonstrated that the mutant VdPEL1rec lacking the enzymatic activity lacked functions to induce both cell death and plant resistance, implying that the enzymatic activity was necessary. In addition, VdPEL1 was strongly induced in V. dahliae infected Nicotiana benthamiana and cotton roots, and VdPEL1 deletion strains severely compromised the virulence of V. dahliae. Our data suggested that VdPEL1 contributed to V. dahliae virulence and induced plant defense responses. These findings provide a new insight for the function of pectate lyase in the host–pathogen interaction.

Botrytis cinerea is one of the most notorious pathogenic species that causes serious plant diseases and substantial losses in agriculture throughout the world. We identified BcXyl1 from B. cinerea that exhibited xylanase activity. Expression of the BcXyl1 gene was strongly induced in B. cinerea infecting Nicotiana benthamiana and tomato plants, and BcXyl1 deletion strains severely compromised the virulence of B. cinerea. BcXyl1 induced strong cell death in several plants, and cell death activity of BcXyl1 was independent of its xylanase activity. Purified BcXyl1 triggered typically PAMP-triggered immunity (PTI) responses and conferred resistance to B. cinerea and TMV in tobacco and tomato plants. A 26-amino acid peptide of BcXyl1 was sufficient for elicitor function. Furthermore, the BcXyl1 death-inducing signal was mediated by the plant LRR receptor-like kinases (RLKs) BAK1 and SOBIR1. Our data suggested that BcXyl1 contributed to B. cinerea virulence and induced plant defense responses.

Background BcGs1, a cell wall-degrading enzyme (CWDE), was originally derived from Botrytis cinerea. Our previous study revealed that BcGs1 could trigger defense responses and protect plants against various pathogens. We researched the defense response mechanism underlying this BcGs1 elicitation in tomato. Results We revealed that the two domains were required for BcGs1’s full necrosis activity. According to analysis and quantitative real-time PCR of the up-regulated proteins and genes filtered by iTRAQ-based quantitative proteome approach, oxidative metabolism and phenylpropanoid metabolism were speculated to be involved in BcGs1-triggered defense response in tomato. Furthermore, experimental evidence showed that BcGs1 triggered reactive oxygen species (ROS) burst and increased the level of phenylalanine-ammonia lyase (PAL) and peroxidase (POD) enzyme activity, as well as lignin accumulation. Moreover, histochemical analysis revealed that infiltration of BcGs1 in tomato leaves exhibited cell wall thickening compared with untreated plants. Conclusions The results suggested that BcGs1 activated the basal defense response included lignin metabolism contributed to BcGs1-induced resistance to Botrytis. cinerea infection in tomato. Electronic supplementary material The online version of this article (10.1186/s12870-018-1319-0) contains supplementary material, which is available to authorized users.

PevD1 is a fungal protein secreted by Verticillium dahliae. Our previous researches showed that this protein could induce hypersensitive responses-like necrosis and systemic acquired resistance (SAR) in cotton and tobacco. To understand immune activation mechanisms whereby PevD1 elicits defense response, the yeast two-hybrid (Y2H) assay was performed to explore interacting protein of PevD1 in Arabidopsis thaliana, and a partner AtNRP (At5g42050) was identified. Here, AtNRP homolog in Nicotiana benthamiana was identified and designated as Nbnrp1. The Nbnrp1 could interact with PevD1 via Y2H and bimolecular fluorescence complementation (BiFC) analyses. Moreover, truncated protein binding assays demonstrated that the C-terminal 132 amino acid (development and cell death, DCD domain) of Nbnrp1 is required for PevD1-Nbnrp1 interaction. To further investigate the roles of Nbnrp1 in PevD1-induced defense response, Nbnrp1-overexpressing and Nbnrp1-silence transgenic plants were generated. The overexpression of Nbnrp1 conferred enhancement of PevD1-induced necrosis activity and disease resistance against tobacco mosaic virus (TMV), bacterial pathogen Pseudomonas syringae pv. tabaci and fungal pathogen V. dahliae. By contrast, Nbnrp1-silence lines displayed attenuated defense response compared with the wild-type. It is the first report that an asparagine-rich protein Nbnrp1 positively regulated V. dahliae secretory protein PevD1-induced cell death response and disease resistance in N. benthamiana.

We report the identification, characterization, and gene cloning of a novel protein elicitor (PeBL1) secreted from Brevibacillus laterosporus strain A60. Through a purification process consisting of ion-exchange chromatography and high-performance liquid chromatography (HPLC), we isolated a protein that was identified by electrospray ionization quadrupole time of flight tandem mass spectrometry (ESI–Q-TOF–MS-MS). The 351-bp PeBL1 gene produces a 12,833-Da protein with 116 amino acids that contains a 30-residue signal peptide. The PeBL1 protein was expressed in Escherichia coli . The recombinant protein can induce a typical hypersensitive response (HR) and systemic resistance in Nicotiana benthamiana , like the endogenous protein. PeBL1-treated N. benthamiana exhibited strong resistance to the infection of tobacco mosaic virus-green fluorescent protein (TMV-GFP) and Pseudomonas syringae pv. tabaci compared to control N. benthamiana . In addition, PeBL1 triggered a cascade of events that resulted in defense responses in plants, including reactive oxygen species (ROS) production, extracellular-medium alkalization, phenolic-compound deposition, and expression of several defense-related genes. Real-time quantitative-PCR analysis indicated that the known defense-related genes PR-1 , PR-5 , PDF1.2 , NPR1 , and PAL were upregulated to varying degrees by PeBL1. This research not only provides insights into the mechanism by which beneficial bacteria activate plant systemic resistance, but also sheds new light on a novel strategy for biocontrol using strain A60.

Abiotic stresses, especially drought, seriously threaten cereal crops yields and quality. In this study, we observed that the rice plants of overexpression the Alternariatenuissima PeaT1 gene showed enhanced drought stress tolerance and increased the survival rate following a drought treatment. In PeaT1-overexpressing (PeaT1OE) plants, abscisic acid (ABA) and chlorophyll content significantly increased, while the malondialdehyde (MDA) content decreased compared with the wild-type plants. Additionally, we confirmed that the transcript levels of drought-responsive genes, including OsAM1, OsLP2, and OsDST, were prominently lower in the PeaT1OE plants. In contrast, expression levels of genes encoding positive drought stress regulators including OsSKIPa, OsCPK9, OsNAC9, OSEREBP1, and OsTPKb were upregulated in PeaT1OE plants. Furthermore, combing the yeast two-hybrid assay, we found that PeaT1 could interact with amyo-inositol oxygenase (OsMIOX), which was verified by pull-down assay. Interestingly, OsMIOX was highly expressed in PeaT1OE plants during the drought treatment. Additionally, the OsMIOX-GFP fusion protein co-localized with the endoplasmic reticulum (ER) marker in tobacco protoplasts, suggesting OsMIOX performs its function in ER. Therefore, our results are useful for elucidating the molecular mechanism underlying the improvement of drought tolerance by PeaT1.

Abiotic stresses, especially drought, seriously threaten cereal crops yields and quality. In this study, we observed that the rice plants of overexpression the Alternariatenuissima PeaT1 gene showed enhanced drought stress tolerance and increased the survival rate following a drought treatment. In PeaT1-overexpressing (PeaT1OE) plants, abscisic acid and chlorophyll content significantly increased, while the malondialdehyde (MDA) content decreased compared with the wild-type plants. Additionally, we confirmed that the transcript levels of drought-responsive genes, including OsAM1, OsLP2, and OsDST, were prominently lower in the PeaT1OE plants. In contrast, expression levels of genes encoding positive drought stress regulators including OsSKIPa, OsCPK9, OsNAC9, OSEREBP1, and OsTPKb were upregulated in PeaT1OE plants. Furthermore, combing the yeast two-hybrid assay, we found that PeaT1 could interact with amyo-inositol oxygenase (OsMIOX), which was verified by pull-down assay. Interestingly, OsMIOX was highly expressed in PeaT1OE plants during the drought treatment. Additionally, the OsMIOX-GFP fusion protein co-localized with the endoplasmic reticulum (ER) marker in tobacco protoplasts, suggesting OsMIOX performs its function in ER. Therefore, our results are useful for elucidating the molecular mechanism underlying the improvement of drought tolerance by PeaT1.

In our previous study, PevD1 was characterized as a novel protein elicitor produced by Verticillium dahliae inducing hypersensitive response (HR) and systemic acquired resistance (SAR) in tobacco plants; however, the detailed mechanisms of PevD1's elicitor activity remain unclear. In this study, five mutant fragments of PevD1 were generated by polymerase chain reaction-based mutagenesis and the truncated proteins expressed in Escherichia coli were used to test their elicitor activities. Biological activity analysis showed that the N-terminal and C-terminal of PevD1 had distinct influence on HR and SAR elicitation. Fragment PevD1ΔN98, which spans the C-terminal 57 amino acids of PevD1, was critical for the induction of HR in tobacco plants. In contrast, fragment PevD1ΔC57, the N-terminal of 98 amino acids of PevD1, retained the ability to induce SAR against tobacco mosaic virus (TMV) but not induction of HR, suggesting that the induction of HR is not essential for SAR mediated by PevD1. Our results indicated that fragment PevD1ΔC57 could be a candidate peptide for plant protection against pathogens without causing negative effects.

We previously identified a novel protein elicitor, PebC1, from Botrytis cinerea and described its enhancement of plant growth, drought tolerance and disease resistance in tomato. Here, we have investigated the defense-associated molecular responses in Arabidopsis thaliana after treatment with recombinant PebC1. PebC1 was expressed in Escherichia coli. Recombinant protein treatments improved plant resistance to Botrytis infection and maintained plant defenses for more than 21 days. The purified protein at 10 μg ml(-1) activated extracellular medium alkalization (pH) and reactive oxygen species and nitric oxide generation and also induced defense gene expression. Arabidopsis mutants that are insensitive to salicylic acid had increased resistance to Botrytis infection after PebC1 treatment but PebC1 did not affect the resistance of mutants with jasmonic acid and ethylene transduction pathways. The results suggest that PebC1 can function as an activator of plant disease resistance and can promote disease resistance to Botrytis in A. thaliana through the ethylene signal transduction pathway.

Elicitors are biofactors that induce resistance in plants against different insect pests. This in vitro study evaluated the impact of a novel elicitor protein PeBC1, extracted from a necrotrophic fungus Botrytis cinerea, on the development and fecundity parameters of green peach aphid (Myzus persicae) on common beans (Phaseolus vulgaris L.). Three different concentrations of PeBC1 elicitor (i.e., 33.56, 25.43, 19.33 &micro, g mL&minus, 1) were applied at three different temperature regimes (i.e., 18, 21, and 25 &deg, C). Elicitor treatments were applied topically on the bean plants at 3-leaf stage and newly emerged (0&ndash, 6 h old) apterous adult aphids were exposed to these treated leaves. In addition to the biological parameters of aphids, the relative expression levels of key genes associated with jasmonic acid (JA) and salicylic acid (SA) plant defense pathways were also determined through RT-qPCR. Results of bioassays revealed that the application of PeBC1 elicitor protein exhibited pronounced and significant (p &lt, 0.05) sub-lethal effects on green peach aphids. The fecundity was reduced and the nymphal development time was prolonged by different concentrations of PeBC1 elicitor and temperature regimes. Gene expression studies showed that the exogenous application of PeBC1 induced a significant upregulation of the expression levels of JA and SA pathway-associated genes in bean plants. As compared to control, elicitor-treated plants exhibited an induced resistance against aphids. Our findings suggest the potential use of PeBC1 elicitor protein in future bio-intensive management strategies against sap-sucking insect pests such as green peach aphids.

A double-stranded (ds) RNA element, sized at approximately 13 kb pairs, was purified from a field isolate, HN10, of Fusarium graminearum. The coding strand of the dsRNA was 13,023 nucleotides (nt) long (excluding the 3' poly(A) tail) and was predicted to contain two discontiguous open reading frames (ORF A and ORF B). The 5' proximal ORF A of 531 nt encoded a protein of 176 amino acids (aa), and a BLAST search showed it to be similar to the putative papain-like protease domains encoded by Valsa ceratosperma hypovirus 1 (35% identity) and Cryphonectria hypovirus 4 (CHV4) (31% identity). The 3' proximal ORF B of 11,118nt encoded a large polyprotein with three conserved domains, including papain-like protease, RNA-dependent RNA polymerase and RNA helicase domains. The polyprotein shared significant aa identities with CHV1 (32%) and CHV2 (32%). Both the genome organization and phylogenetic analysis suggested that the characterized RNA represented a novel hypovirus, designated "Fusarium graminearum hypovirus 1 (FgHV1)", which was closely related to CHV1 and CHV2 in the Hypoviridae family. Elimination of the virus resulted in no dramatic phenotypic alteration of the fungus.

RNA interference (RNAi) caused by exogenous double-stranded RNA (dsRNA) has developed into a powerful technique in functional genomics, and to date it is widely used to down-regulate crucial physiology-related genes to control pest insects. A molt-regulating transcription factor gene, HaHR3, of cotton bollworm (Helicoverpa armigera) was selected as the target gene. Four different fragments covering the coding sequence (CDS) of HaHR3 were cloned into vector L4440 to express dsRNAs in Escherichia coli. The most effective silencing fragment was then cloned into a plant over-expression vector to express a hairpin RNA (hpRNA) in transgenic tobacco (Nicotiana tabacum). When H. armigera larvae were fed the E. coli or transgenic plants, the HaHR3 mRNA and protein levels dramatically decreased, resulting developmental deformity and larval lethality. The results demonstrate that both recombinant bacteria and transgenic plants could induce HaHR3 silence to disrupt H. armigera development, transgenic plant-mediated RNAi is emerging as a powerful approach for controlling insect pests.

Fusarium graminearum hypovirus 1 (FgHV1), which is phylogenetically related to Cryphonectria hypovirus 1 (CHV1), is a virus in the family Hypoviridae that infects the plant pathogenic fungus F. graminearum. Although hypovirus FgHV1 infection does not attenuate the virulence of the host (hypovirulence), it results in defects in mycelial growth and spore production. We now report that the vertical transmission rate of FgHV1 through asexual spores reached 100%. Using RNA deep sequencing, we performed genome-wide expression analysis to reveal phenotype-related genes with expression changes in response to FgHV1 infection. A total of 378 genes were differentially expressed, suggesting that hypovirus infection causes a significant alteration of fungal gene expression. Nearly two times as many genes were up-regulated as were down-regulated. A differentially expressed gene enrichment analysis identified a number of important pathways. Metabolic processes, the ubiquitination system, and especially cellular redox regulation were the most affected categories in F. graminearum challenged with FgHV1. The p20, encoded by FgHV1 could induce H2O2 accumulation and hypersensitive response in Nicotiana benthamiana leaves. Moreover, hypovirus FgHV1 may regulate transcription factors and trigger the RNA silencing pathway in F. graminearum.

Previously, we characterized F. graminearum hypovirus 1 (FgHV1) and F. graminearum hypovirus 2 (FgHV2), which are the only two hypoviruses in F. graminearum that are closely related to Cryphonectria hypovirus 1 (CHV1) and Cryphonectria hypovirus 2 (CHV2) in the Hypoviridae family. In this study, we preliminarily elucidated the RNA silencing mechanism of the F. graminearum/hypovirus system from a small RNA (sRNA) perspective by using HiSeq deep sequencing. The length distributions of F. graminearum sRNA were altered by hypoviral infection. Potential microRNA-like (milRNA) candidates were differentially expressed between the hypovirus-free and hypovirus-infected library types. Extensive virus-derived small interfering RNAs (vsiRNAs) were also principally defined. The 1,831,081 and 3,254,758 total reads generated from the FgHV1 and FgHV2 genomes in F. graminearum yielded the first high-resolution sRNA maps of fungal viruses. In addition, extensive bioinformatics searches identified a large number of transcripts that are potentially targeted by vsiRNAs, several of which were effectively down-regulated. In particular, the RNA silencing-related genes FgDicer1 and FgRdRp5 were predicted targets of FgHV1- and FgHV2-derived siRNAs, possibly revealing a novel anti-RNA silencing strategy employed by mycoviruses.

Symbiotic bacteria, which are carried in the intestinal vesicle of the infective stage of juvenile entomopathogenic nematodes, produce broad-spectrum antibiotics. In this study, we aimed to isolate the antimicrobial peptides from the culture of the entomopathogenic bacterium Xenorhabdus budapestensis NMC-10. By screening chromatography columns and optimizing flow rate, pH, salinity and other purification conditions, we identified the final purification procedures which consisted of Q ion-exchange chromatography, gel filtration chromatography and two-step reverse-phase chromatography. Two novel antimicrobial peptides were identified via Q-TOF-TOF and de novo sequencing, and designated as GP-19 and EP-20. Both natural and synthetic peptides demonstrated broad-spectrum antimicrobial activities. The synthetic GP-19 peptide was active against Verticillium dahlia with EC50 values of 17.54 μg/ml and highly inhibited the growth of a variety of bacteria, while the synthetic EP-20 peptide was highly active against Phytophthora capsici with EC50 values of 3.14 μg/ml.

Potato late blight disease, which is caused by the fungus Phytophthora infestans, results in considerable loss of potato crop yield worldwide. Developing new bio-agents to control this disease is desirable. Xenocoumacin 1 (Xcn1) is an antibacterial substance from the entomopathogenic nematode symbiotic bacterium, Xenorhabdus nematophila var. pekingensis. In this study, we evaluated the antifungal activity of Xcn1, along with its potential activity against Phytophthora infestans, in vitro and in vivo. The results showed that Xcn1 exhibits strong antifungal activity against five species of Phytophthora, with EC50 values ranging from 0.25 to 4.17 μg/mL. Xcn1 not only inhibited mycelial growth of P. infestans, reaching 100% inhibition at 1.5 μg/mL of Xcn1, but also suppressed sporangia production. Xcn1 also showed potent in vivo activity against P. infestans, with 92.63% and 80.27% in detached plants and potted plants, respectively, in comparison with the control. Therefore, Xcn1 has antibiotic activities against P. infestans both in vitro and in vivo.

Xenorhabdus nematophila var. pekingensis, which is highly virulent for many insects, is a symbiotic bacterium of Steinernema carpocapsae isolated from Beijing soil in China. Previous studies demonstrated that the bacterium had high antifeedant activity against larvae of Helicoverpa armigera, Plutella xylostella and Spodoptera exigua. Herein, we report the purification, molecular cloning and antifeedant activity of an intracellular toxic protein from the bacterium. The purified protein displayed a single band and a relative molecular weight of over 212 kDa determined by SDS-PAGE. We designated the protein as XnAFP2. Peptide segments were obtained by MALDI-TOF and covered 40% of the amino acid sequence of a toxin protein from X. nematophilus PMFI1296. The full cDNA sequence encoding for XnAFP2 (Genbank accession number FJ222606) was amplified from X. nematophlia var. pekingensis and consists of 7575 bp. The gene showed homology with up to 99% identity to the A2 gene from X. nematophila strain BP (G...

Elicitors are molecules that stimulate defense responses in plants. Previously, an elicitor-encoding gene, named pemG1, was isolated from Magnaporthe grisea. To assess the function of pemG1 in rice (Oryza sativa L. cv. Nipponbare), the gene was cloned under a constitutive maize ubiquitin promoter and introduced into Nipponbare cultivar. The resultant plants showed stable integration and constitutive expression of the pemG1 gene. The expression of defense-related gene for phenylalanine ammonia-lyase was triggered and proline content was also increased in pemG1-expressing plants. The pemG1-expressing plants showed enhanced resistance against rice blast after inoculation with M. grisea spores, suggesting that the pemG1 expression enhances disease resistance in transgenic rice.

Alternaria tenuissima is a fungus widely present in the environment and could cause diseases in plants and humans. In this study, through a yeast genetic approach, cDNA sequences were isolated and characterized for the AtHIS3 and AtLEU2 genes. AtHIS3 cDNA encodes a protein of 238 amino acids, while AtLEU2 cDNA encodes a protein of 363 amino acids. Based on the phylogenetic analysis of amino acid sequences of AtHis3p and AtLeu2p, A. tenuissima is closely related to the plant pathogenic fungus Phaeosphaeria nodorum. This study provides two genetic markers for studies of functions of genes regulating development, morphology, and virulence of A. tenuissima.

Plant-specific NAC transcription factors (TFs) constitute a large family and play important roles in regulating plant developmental processes and responses to environmental stresses, but only some of them have been investigated for effects on disease reaction in cereal crops. Virus-induced gene silencing (VIGS) is an effective strategy for rapid functional analysis of genes in plant tissues. In this study, TaNAC1, encoding a new member of the NAC1 subgroup, was cloned from bread wheat and characterized. It is a TF localized in the cell nucleus, and contains an activation domain in its C-terminal. TaNAC1 was strongly expressed in wheat roots and was involved in responses to infection by the obligate pathogen Puccinia striiformis f. sp. tritici and defense-related hormone treatments such as salicylic acid (SA), methyl jasmonate, and ethylene. Knockdown of TaNAC1 with barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) enhanced stripe rust resistance. TaNAC1-overexpression in Arabidopsis thaliana plants gave enhanced susceptibility, attenuated systemic-acquired resistance to Pseudomonas syringae DC3000, and promoted lateral root development. Jasmonic acid-signaling pathway genes PDF1.2 and ORA59 were constitutively expressed in transgenic plants. TaNAC1 overexpression suppressed the expression levels of resistance-related genes PR1 and PR2 involved in SA signaling and AtWRKY70, which functions as a connection node between the JA- and SA-signaling pathways. Collectively, TaNAC1 is a novel NAC member of the NAC1 subgroup, negatively regulates plant disease resistance, and may modulate plant JA- and SA-signaling defense cascades.

In this study, a necrosis-inducing protein was purified from the culture filtrate of the necrotrophic fungus Botrytis cinerea BC-98 strain. Secreted proteins were collected and fractionated by liquid chromatography. The fraction with the highest necrosis-inducing activity was further purified. A glycoprotein named BcGs1 was identified by 2D electrophoresis and mass spectrometry. The BcGs1 protein consisted of 672 amino acids with a theoretical molecular weight of 70.487 kDa. Functional domain analysis indicated that BcGs1 was a glucan 1,4-alpha-glucosidase, a cell wall-degrading enzyme, with a Glyco_hydro_15 domain and a CBM20_glucoamylase domain. The BcGs1 protein caused necrotic lesions that mimicked a typical hypersensitive response and H2O2 production in tomato and tobacco leaves. BcGs1-treated plants exhibited resistance to B. cinerea, Pseudomonas syringae pv. tomato DC3000 and tobacco mosaic virus in systemic leaves. In addition, BcGs1 triggered elevation of the transcript levels of the defence-related genes PR-1a, TPK1b and Prosystemin. This is the first report of a Botrytis glucan 1,4-alpha-glucosidase triggering host plant immunity as an elicitor. These results lay a foundation for further study of the comprehensive interaction between plants and necrotrophic fungi.

RNA interference (RNAi) has been developed as an efficient technology. RNAi insect-resistant transgenic plants expressing double-stranded RNA (dsRNA) that is ingested into insects to silence target genes can affect the viability of these pests or even lead to their death. HaHR3, a molt-regulating transcription factor gene, was previously selected as a target expressed in bacteria and tobacco plants to control Helicoverpa armigera by RNAi technology. In this work, we selected the dsRNA-HaHR3 fragment to silence HaHR3 in cotton bollworm for plant mediated-RNAi research. A total of 19 transgenic cotton lines expressing HaHR3 were successfully cultivated, and seven generated lines were used to perform feeding bioassays. Transgenic cotton plants expressing dsHaHR3 were shown to induce high larval mortality and deformities of pupation and adult eclosion when used to feed the newly hatched larvae, and 3rd and 5th instar larvae of H. armigera. Moreover, HaHR3 transgenic cotton also demonstrated an improved cotton yield when compared with controls.

Hrip1 is a novel hypersensitive response-inducing protein secreted by Alternaria tenuissima that activates defense responses and systemic acquired resistance in tobacco. This study investigates the role that Hrip1 plays in responses to abiotic and biotic stress using transgenic Arabidopsis thaliana expressing the Hrip1 gene under the control of the stress-inducible rd29A promoter or constitutive cauliflower mosaic virus 35S promoter. Bioassays showed that inducible Hrip1 expression in rd29A∷Hrip1 transgenic lines had a significantly higher effect on plant height, silique length, plant dry weight, seed germination and root length under salt and drought stress compared to expression in 35S∷Hrip1 lines and wild type plants. The level of enhancement of resistance to Botrytis cinerea by the 35S∷Hrip1 lines was higher than in the rd29A∷Hrip1 lines. Moreover, stress-related gene expression in the transgenic Arabidopsis lines was significantly increased by 200 mM NaCl and 200 mM mannitol treatments, and defense genes in the jasmonic acid and ethylene signaling pathway were significantly up-regulated after Botrytis inoculation in the Hrip1 transgenic plants. Furthermore, the activity of some antioxidant enzymes, such as peroxidase and catalase increased after salt and drought stress and Botrytis infection. These results suggested that the Hrip1 protein contributes to abiotic and biotic resistance in transgenic Arabidopsis and may be used as a useful gene for resistance breeding in crops. Although the constitutive expression of Hrip1 is suitable for biotic resistance, inducible Hrip1 expression is more responsive for abiotic resistance.

MoHrip2 Magnaporthe oryzae hypersensitive protein 2 is an elicitor protein of rice blast fungus M. oryzae. Rice seedlings treated with MoHrip2 have shown an induced resistance to rice blast. To elucidate the mechanism underlying this MoHrip2 elicitation in rice, we used differential-display 2-D gel electrophoresis and qRT-PCR to assess the differential expression among the total proteins extracted from rice leaves at 24 h after treatment with MoHrip2 and buffer as a control. Among ~1000 protein spots detected on each gel, 10 proteins were newly induced, 4 were up-regulated, and 3 were down-regulated in MoHrip2-treated samples compared with the buffer control. Seventeen differentially expressed proteins were detected using MS/MS analysis and categorized into six groups according to their putative function: defense-related transcriptional factors, signal transduction-related proteins, reactive oxygen species (ROS) production, programmed cell death (PCD), defense-related proteins, and photosynthesis and energy-related proteins. The qPCR results (relative expression level of genes) further supported the differential expression of proteins in MoHrip2-treated rice leaves identified with 2D-gel, suggesting that MoHrip2 triggers an early defense response in rice leaves via stress-related pathways, and the results provide evidence for elicitor-induced resistance at the protein level.

The bacteria Xenorhabdus spp. are entomopathogenic symbionts that can produce several toxic proteins that interfere with the immune system of insects. Recently, we purified the insecticidal protein XeGroEL from Xenorhabdus ehlersii and discovered that injection of XeGroEL into larvae of Galleria mellonella triggers strong immune responses. In this study, we determined the level of induction of several immune-responsive proteins that were secreted into the hemolymph using comparative proteomic analyses of hemolymph proteins from XeGroEL-challenged larvae. Additionally, quantitative real-time reverse transcription-PCR analyses demonstrated increased transcriptional rates of immune-related genes at 5 h post-challenge with purified XeGroEL. Our results help to understand anti-microbial immune responses in G. mellonella, suggesting that the immune system recognizes exogenous proteins and pathogen-associated molecular patterns.

Myzus persicae is an economically important pest infecting more than 40 families of crops throughout the world. Six entomopathogenic fungal isolates, three each of Beauveria bassiana and Verticillium lecanii , were screened for pathogenicity test against the M. persicae to select high virulent isolate with the most suitable application and to determine the role of individual enzyme in its virulence. Two treatments that is, conidial shower (190±23 conidia/mm 2 ) and filtrate (3 ml filtrate per treatment from six days liquid broth culture of 1.0x10 8 conidia ml -1 ) were conducted for virulence or toxicity test and a comparison was made between treatments and among fungal isolates against the target pest. The percent mortality rates of filtrate at each day, after inoculation was found higher as compared to percent mortality of conidial showering. V. lecanii 3 showed highest virulence or toxicity against the target pest treated either with conidial (80.70%) or filtrate (88.36%) application while B. bassiana 70 and B. bassiana 76 showed high toxicity (77.14 and 80.86%, respectively) in filtrate application at 6 th day of incubation. The aphidicial activities of the fungal isolates were evaluated by correlating the enzymes (Chitinase, protease and lipase) activities with enzymes production. On the basis of enzymatic activities, lipase was assumed to participate more in the total virulence or pathogenicity as compared to protease and chitinase while protease was assumed to participate more than chitinase. The pathogenicity test reveals the selection, effective application of most virulent isolate and the role of individual enzyme to develop an alternative control agent against M. persicae . Keywords: Beauveria bassiana , filtrate, conidia, enzymes, Verticillium lecanii

The effector protein PevD1 from the pathogenic fungusVerticillium dahliaewas purified and crystallized using the hanging-drop vapour-diffusion method. Native crystals appeared in a solution consisting of 4.0 Msodium formate. A native data set was collected at 1.9 Å resolution at 100 K using an in-house X-ray source. Because of the absence of useful methinione in the protein sequence, derivative crystals that contained iodine were obtained by soaking in 1.25 Mpotassium iodide, and a data set that contained anomalous signal was collected using the same X-ray facility at a wavelength of 1.54 Å. The single-wavelength anomalous dispersion method was used to successfully solve the structure based on the anomalous signal generated from iodine.

The bacteria Xenorhabdus spp. are entomopathogenic symbionts that can produce several toxic proteins that interfere the immune system of insects. We purified an insecticidal protein from Xenorhabdus ehlersii, and designated it as XeGroEL with an estimated molecular mass of ~58 kDa. Galleria mellonella larva injected with XeGroEL presented prophenoloxidase activation and hemocyte decrease. XeGroEL can kill G. mellonella larva in 48 h with an LD(50) of 0.76 ± 0.08 μg/larva. Our results demonstrate that X. ehlersii possesses a toxic XeGroEL protein acting as a potential factor to activate proPO in host insect, which also provides a meaningful hypothesis to understand the interaction between nematode-symbiotic bacteria and host.

An attractive alternative method to chemical pesticides is the microbial biocontrol (MBCAs) agents. They are the natural enemies devastating the pest population with no hazard effects on human health and the environment. Entomopathogenic fungi has an important position among all the biocontrol agents because of its route of pathogenicity, broad host rang and its ability to control both sap sucking pests such as mosquitoes and aphids as well as pests with chewing mouthparts, yet they only cover a small percentage of the total insecticide market. Improvements are needed to fulfill the requirements for high market share. Entomopathogenic fungi differ from other microorganisms in their infection process: they directly breach the cuticle to enter the insect hemocoel, while other microorganisms enter by ingestion through mouth and then cause disease. Insect cuticle is mainly composed of chitin and protein surrounded by wax, lipid layer or fatty acids. Fungal pathogenesis mainly starts with the secretion of cuticle degrading enzymes. Some important and well known cuticle degrading enzymes are chitinase, protease and lipase which can degrade chitin, protein and lipid of the cuticle, respectively. In this review we collected literatures from different sources and we arranged them in a such a way to better to understand the process of insect pathogenicity of entomopathogenic fungi and to find ways to improve the virulence of wild strain fungi to shorten the killing time of the pest and to commercialize the entomopathogenic fungi. In this way, the market share of the fungal entomopathogenic fungi will increase and a decrease in the usage of synthetic chemical pesticides will automatically follow.

A novel antimicrobial peptide, with molecular mass of 1602.0469Da, produced by Brevibacillus laterosporus strain A60 was isolated and purified from the soil of mango plants. The purification procedure consisted of ammonium sulfate precipitation, cation exchange chromatography on an HiTrap SP HP column, thin layer chromatography and High Performance Liquid Chromatography (HPLC) on C18 reversed-phase column. After the four isolation procedures, one peptide with antimicrobial activity was obtained and named BL-A60. The determination of the complete amino acid sequences of this peptide showed that it contains eleven amino acid residues, L-Y-K-L-V-K-V-V-L-N-M, and a choline connected to the N-terminal and a tenuazonic acid modified of the C-terminal. This peptide shows relatively low identification to other antimicrobial peptides from bacteria. Purified BL-A60 showed high pH and thermal stability and a strong inhibition of different stages of the life cycle of Phytophthora capsici, including mycelial growth, sporangia formation and cystospore germination, with EC(50) values of 7.89, 0.60 and 21.96 μg ml(-1), respectively.

Xenorhabdus budapestensis can produce a variety of proteins that help this bacterium and its mutualistic nematode vector kill the host insect. In this report, we purified one protein fraction from the intracellular extract of X. budapestensis D43, which was designated HIP57. By injection, HIP57 caused Galleria mellonella larval bodies to blacken and die with an LD(50) of 206.81 ng/larva. Analyzes of HIP57 by two-dimensional gel electrophoresis showed that this protein was a single spot on the gel with a molecular weight of 57 kDa and a pI of ∼5. Sequencing and bioinformatic analysis suggested that the HIP57 toxin was homologous to GroEL. GroEL has been accepted as molecule chaperon; however, our research revealed that HIP57 (GroEL) possesses another novel function as an insecticide. A GroEL phylogenetic tree defined the relationship among the related species of mutualistic bacteria (Xenorhabdus and Photorhabdus) from the entomopathogenic nematodes and the evolution within the family Enterobacteriaceae. Thus, GroEL could be a complement to 16S rDNA for studying the molecular phylogenies of the family Enterobacteriaceae. Phenoloxidase (PO) activity analysis of G. mellonella larvae injected with HIP57 suggested that the toxin activates the PO cascade, which provides an extensive defense reaction that potentially responsible for G. mellonella larval death.