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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. [ABSTRACT FROM AUTHOR]

The soil-borne fungal pathogen Verticillium dahliae infects a wide range of dicotyledonous plants including cotton, tobacco, and Arabidopsis. Among the effector proteins secreted by V. dahliae, the 16 kDa PevD1 induces a hypersensitive response in tobacco. Here we report the high-resolution structure of PevD1 with folds resembling a C2 domain-like structure with a calcium ion bound to the C-terminal acidic pocket. A yeast two-hybrid screen, designed to probe for molecular functions of PevD1, identified Arabidopsis asparagine-rich protein (NRP) as the interacting partner of PevD1. Extending the pathway of V. dahliae effects, which include induction of early flowering in cotton and Arabidopsis, NRP was found to interact with cryptochrome 2 (CRY2), leading to increased cytoplasmic accumulation of CRY2 in a blue light-independent manner. Further physiological and genetic evidence suggests that PevD1 indirectly activates CRY2 by antagonizing NRP functions. The promotion of CRY2-mediated flowering by a fungal effector outlines a novel pathway by which an external stimulus is recognized and transferred in changing a developmental program. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Two novel double-stranded RNA (dsRNA) mycoviruses, termed Fusarium poae dsRNA virus 2 (FpV2) and Fusarium poae dsRNA virus 3 (FpV3), were isolated from the plant pathogenic fungus, Fusarium poae strain SX63, and molecularly characterized. FpV2 and FpV3, with respective genome sequences of 9518 and 9419 base pairs (bps), are both predicted to contain two discontinuous open reading frames (ORFs), ORF1 and ORF2. A hypothetical polypeptide (P1) and a RNA-dependent RNA polymerase (RdRp) are encoded by ORF1 and ORF2, respectively. Phytoreo_S7 domain (pfam07236) homologs were detected downstream of the RdRp domain (RdRp_4; pfam02123) of the ORF2-coded proteins of both FpV2 and FpV3. The same shifty heptamers (GGAAAAC) were both found immediately before the stop codon UAG of ORF1 in FpV2 and FpV3, which could mediate programmed -1 ribosomal frameshifting (-1 PRF). Phylogenetic analysis based on RdRp sequences clearly place FpV2 and FpV3 in a taxonomically unassigned dsRNA mycovirus group. Together, with a comparison of genome organization, a new taxonomic family termed Fusagraviridae is proposed to be created to include FpV2- and FpV3-related dsRNA mycoviruses, within which FpV2 and FpV3 would represent two distinct virus species. [ABSTRACT FROM AUTHOR]

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 SAsignaling 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. [ABSTRACT FROM AUTHOR]

Systemic acquired resistance (SAR) is an inducible defense mechanism which plays a central role in protecting plants from pathogen attack. A new elicitor, PeaT1 from Alternaria tenuissima, was expressed in Escherichia coil and characterized with systemic acquired resistance to tobacco mosaic virus (TMV). PeaT1-treated plants exhibited enhanced systemic resistance with a significant reduction in number and size of TMV lesions on wild tobacco leaves as compared with control. The quantitative analysis of TMV CP gene expression with real-time quantitative PCR showed there was reduction in TMV virus concentration after PeaT1 treatment. Similarly, peroxidase (POD) activity and lignin increased significantly after PeaT1 treatment. The real-time quantitative PCR revealed that PeaT1 also induced the systemic accumulation of pathogenesis-related gene, PR- 1a and PR- 1b which are the markers of systemic acquired resistance (SAR), NPR1 gene for salicylic acid (SA) signal transduction pathway and PAL gene for SA synthesis. The accumulation of SA and the failure in development of similar level of resistance as in wild type tobacco plants in PeaT1 treated nahG transgenic tobacco plants indicated that PeaT1-induced resistance depended on SA accumulation. The present work suggested that the molecular mechanism of PeaT1 inducing disease resistance in tobacco was likely through the systemic acquired resistance pathway mediated by salicylic acid and the NPR1 gene. [ABSTRACT FROM AUTHOR]

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 EC 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. [ABSTRACT FROM AUTHOR]

Both mammalian tensin-like phosphatase 1 [TEP1; also known as phosphatase deleted on chromosome 10 (PTEN) or mutated in multiple advanced cancer 1 (MMAC1)] and Saccharomyces cerevisiae ScTep1p are involved in the phosphatidylinositol pathway. In this study, we identified the Fusarium graminearum locus FGSG_04982.3 (named FgTEP1) as the functional homologue of ScTEP1 in the sensitivity of S. cerevisiae cells to wortmannin, the phosphatidylinositol-3 kinase inhibitor. Deletion of FgTEP1 causes F. graminearum mycelial growth to become sensitive to lithium and reduces the production of conidia. Although conidia lacking FgTEP1 germinate normally, they show reduced germination efficiency in the presence of wortmannin. In addition, we showed that deletion of FgTEP1 reduces the virulence of F. graminearum on wheat. These results indicate that FgTep1p is linked to the phosphatidylinositol-3 kinase signalling pathway in this plant fungal pathogen. [ABSTRACT FROM AUTHOR]

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 (GenBank accession number AY282763) and 92% identity to the insecticidal toxin xptA2 gene from X. nematophila PMFI 1296 (GenBank accession number AJ308438). The protein caused a rapid cessation in feeding and reduction in larval weight of H. armigera. When fed to third instar larvae of H. armigera in an artificial diet at 6.0 µg/g (w/w) toxin protein, growth reduction reached 97.9%. The insecticidal protein greatly decreased fourth instar larval weight, lengthened larval stage, and reduced pupation and emergence rates. The antifeedant rate in choice and no-choice leaf disk tests against fifth instar larvae was 78.4 and 87.6% in 24 h, respectively. [ABSTRACT FROM AUTHOR]

Beauveria brongniartii extracellular subtilisin-like serine protease (Pr1) is one of the most virulent factors by virtue of its activity against insect cuticles. The Pr1 cDNA was cloned using the switching mechanism at the 5′ end of the RNA transcript and rapid amplification of cDNA ends. The 1732-bp fragment of genomic DNA containing the predicted open-reading frame of the Pr1 gene was cloned by polymerase chain reaction and sequenced. The Pr1 cDNA is 1550 bp and contains an 1140-bp ORF. The deduced amino-acid sequence of the protein shows identity to that of proteinase K from Tritirachium album (62%), Pr1 from Metarhizium nisopliae (67%), and Pr1 from B. bassiana (76%). The Pr1 protein with an N-terminal fusion to the six-histidine tag was expressed in Escherichia coli as inclusion bodies with the expression vector pBV220. Sodium dodecylsulsulfate–polyacrylamide gel electrophoresis clearly revealed expressed product. The Pr1 protein was purified and refolded and had proteolytic activity of 0.288 U mg−1. [ABSTRACT FROM AUTHOR]

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 ℃). 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) pathwayassociated 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. [ABSTRACT FROM AUTHOR]

Objective: We constructed cDNA library of Magnaporthe grisea. The good quality cDNA library could facilitate finding proteinaceous elicitors of M. grisea, and elucidating the mechanisms of the M. grisea--rice interaction., Methods: The Oligo(dT) combined with the magnetic bead was used to extract mRNA from total RNA of Magnaporthe grisea and as primers to synthesize the first-strand cDNA. Terminal transferase introduced PolyA into 3'terminal of the first cDNA strand, then the PolyA was used for amplifying the second-strand cDNA. Restriction enzyme and adapter were avoided in this research, which could solve technical limitation of the traditional method. Because all reactions were done in one centrifuge tube, this process could reduce the risk of cDNA loss and cross-contamination. The primers designed in this research could clone the amplified cDNAs into expression vector in a desirable orientation., Results: The cDNA library constructed had a high titer of 8.9 x 10(6) cfu/mL, and contained a total clones of 8.9 x 10(7) cfu, with an average inserts size of about 1380 bp., Conclusion: Constructing cDNA library with magnetic bead was a highly efficient method using only small amount of experimental materials within a short period.