Differential activation of defense-related genes in susceptible and resistant pepper cultivars infected with Phytophthora capsici

This study investigated the expression pattern of genes encoding for a basic PR-1 protein, a basic beta-1,3-glucanase, a peroxidase, and a sesquiterpene cyclase involved in defense responses in three pepper cultivars with different levels of resistance to Phytophthora capsici. All genes were up-regulated in infected stems of the pepper cultivars, with expression being detected 8h post-inoculation. mRNA levels of these genes increased markedly by 24h post-inoculation, and maximal induction levels were observed for the PR-1 and sesquiterpene cyclase genes. PR-1, peroxidase, and sesquiterpene genes were always expressed at higher levels in resistant cultivars than in the susceptible cultivar, although up-regulation was observed in both, suggesting that the differences between these pepper genotypes in susceptibility and resistance are a matter of the timing and magnitude of the defense response.     

Recovery of transgenic rice plants expressing the rice dwarf virus outer coat protein gene (S8)

 The coding region of the eighth largest segment (S8) of the rice dwarf virus (RDV) was obtained from a RDV Fujian isolate. It was then cloned into pTrcHisA for expression in E. coli and into vector pE3 for plant transformation. By using callus derived from mature rice embryos as the target tissue, we obtained regenerated rice plants after bombardment of the former with plasmid pE3R8 containing the RDV S8 gene and the marker gene neomycin phosphotransferase (NPT II). Southern blotting confirmed the integration of the RDV S8 gene into the rice genome. The expression of the outer coat protein in both E. coli and rice plants was confirmed by western blotting. The recovery of transgenic rice plants expressing S8 gene is an important step towards studying the function of the RDV genes and obtaining RDV-resistant rice plants. Received: 1 March 1996 / Accepted: 2 August 1996     

Involvement of Trichoderma trichothecenes in the biocontrol activity and induction of plant defense-related genes

Trichoderma species produce trichothecenes, most notably trichodermin and harzianum A (HA), by a biosynthetic pathway in which several of the involved proteins have significant differences in functionality compared to their Fusarium orthologues. In addition, the genes encoding these proteins show a genomic organization differing from that of the Fusarium tri clusters. Here we describe the isolation of Trichoderma arundinaceum IBT 40837 transformants which have a disrupted or silenced tri4 gene, a gene encoding a cytochrome P450 monooxygenase that oxygenates trichodiene to give rise to isotrichodiol, and the effect of tri4 gene disruption and silencing on the expression of other tri genes. Our results indicate that the tri4 gene disruption resulted in a reduced antifungal activity against Botrytis cinerea and Rhizoctonia solani and also in a reduced ability to induce the expression of tomato plant defense-related genes belonging to the salicylic acid (SA) and jasmonate (JA) pathways against B. cinerea, in comparison to the wild-type strain, indicating that HA plays an important function in the sensitization of Trichoderma-pretreated plants against this fungal pathogen. Additionally, the effect of the interaction of T. arundinaceum with B. cinerea or R. solani and with tomato seedlings on the expressions of the tri genes was studied.     

Wheat dwarf virus vectors replicate and express foreign genes in cells of monocotyledonous plants.

Wheat dwarf virus (WDV) is a geminivirus that infects monocotyledonous plants. To exploit the potential of WDV as a replicative gene vector, we developed a transient replication and expression system based on the transfection of protoplasts derived from Triticum monococcum suspension culture cells. Cloned genomic copies of various WDV isolates as well as mutants constructed in vitro were introduced into the protoplasts and assayed for their ability to replicate. As a result, regions of the WDV genome necessary or dispensable for the viral DNA replication could be defined. In addition, the gene encoding the viral capsid protein was replaced by three different bacterial marker genes, neomycin phosphotransferase, chloramphenicol acetyltransferase, and beta-galactosidase. The beta-galactosidase gene doubled the size of the WDV genome. The replication of the recombinant WDV genomes and the expression of these genes were monitored in suspension culture cells of T. monococcum. The potential of replicative expression vectors based on the WDV genome is discussed.     

Assembly of double-shelled, virus-like particles in transgenic rice plants expressing two major structural proteins of rice dwarf virus

Rice dwarf virus (RDV) is a double-shelled particle that contains a major capsid protein (P8), a major core protein (P3), several minor core proteins, and viral genomic double-stranded RNA. Coexpression of P8 and P3 in transgenic rice plants resulted in formation of double-shelled, virus-like particles (VLPs) similar to the authentic RDV particles. The VLPs were not detected in transgenic rice plant cells expressing P8 alone. This in vivo result suggests that P8 interacted with P3 and that these two proteins provide the structural integrity required for the formation of VLPs in rice cells independently of other structural proteins, nonstructural proteins, or viral genomic double-stranded RNAs.     

Suppression of auxin signalling promotes rice susceptibility to Rice black streaked dwarf virus infection

Auxin plays a fundamental role in plant growth and development, and also influences plant defence against various pathogens. Previous studies have examined the different roles of the auxin pathway during infection by biotrophic bacteria and necrotrophic fungi. We now show that the auxin signalling pathway was markedly down-regulated following infection of rice by Rice black streaked dwarf virus (RBSDV), a dsRNA virus. Repression of the auxin receptor TIR1 by a mutant overexpressing miR393 increased rice susceptibility to RBSDV. Mutants overexpressing the auxin signalling repressors OsIAA20 and OsIAA31 were also more susceptible to RBSDV. The induction of jasmonic acid (JA) pathway genes in response to RBSDV was supressed in auxin signalling mutants, suggesting that activation of the JA pathway may be part of the auxin signalling-mediated rice defence against RBSDV. More importantly, our results also revealed that OsRboh-mediated reactive oxygen species levels played important roles in this defence. The results offer novel insights into the regulatory mechanisms of auxin signalling in the rice–RBSDV interaction.     

The diphenylether herbicide lactofen induces cell death and expression of defense-related genes in soybean

Lactofen belongs to the diphenylether class of herbicides, which targets protoporphyrinogen oxidase, which in turn causes singlet oxygen generation. In tolerant plants like soybean (Glycine max), the chemical nonetheless causes necrotic patches called "bronzing" in contact areas. Here it is shown that such bronzing is accompanied by cell death, which was quantified from digital microscopic images using Assess Software. Cellular autofluorescence accompanied cell death, and a homolog of the cell death marker gene, Hsr203j, was induced by lactofen in treated soybean tissues. Thus, this form of chemically induced cell death shares some hallmarks of certain types of programmed cell death. In addition to the cell death phenotype, lactofen caused enhanced expressions of chalcone synthase and chalcone reductase genes, mainly in the exposed and immediately adjacent (proximal) cells. Furthermore, isoflavone synthase genes, which are wound inducible in soybean, were up-regulated by lactofen in both proximal and distal cell zones in minimally wounded cotyledons and further enhanced in wounded tissues. Moreover, if the wall glucan elicitor from Phytophthora sojae was present during lactofen treatment, the induction of isoflavone synthase was even more rapid. These results are consistent with the fact that lactofen triggers massive isoflavone accumulations and activates the capacity for glyceollin elicitation competency. In addition, lactofen induces late expression of a selective set of pathogenesis-related (PR) protein genes, including PR-1a, PR-5, and PR-10, mainly in treated proximal tissues. These various results are discussed in the context of singlet oxygen-induced responses and lactofen's potential as a disease resistance-inducing agent.     

The rice dwarf virus P2 protein interacts with ent-kaurene oxidases in vivo, leading to reduced biosynthesis of gibberellins and rice dwarf symptoms

The mechanisms of viral diseases are a major focus of biology. Despite intensive investigations, how a plant virus interacts with host factors to cause diseases remains poorly understood. The Rice dwarf virus (RDV), a member of the genus Phytoreovirus, causes dwarfed growth phenotypes in infected rice (Oryza sativa) plants. The outer capsid protein P2 is essential during RDV infection of insects and thus influences transmission of RDV by the insect vector. However, its role during RDV infection within the rice host is unknown. By yeast two-hybrid and coimmunoprecipitation assays, we report that P2 of RDV interacts with ent-kaurene oxidases, which play a key role in the biosynthesis of plant growth hormones gibberellins, in infected plants. Furthermore, the expression of ent-kaurene oxidases was reduced in the infected plants. The level of endogenous GA1 (a major active gibberellin in rice vegetative tissues) in the RDV-infected plants was lower than that in healthy plants. Exogenous application of GA3 to RDV-infected rice plants restored the normal growth phenotypes. These results provide evidence that the P2 protein of RDV interferes with the function of a cellular factor, through direct physical interactions, that is important for the biosynthesis of a growth hormone leading to symptom expression. In addition, the interaction between P2 and rice ent-kaurene oxidase-like proteins may decrease phytoalexin biosynthesis and make plants more competent for virus replication. Moreover, P2 may provide a novel tool to investigate the regulation of GA metabolism for plant growth and development.     

Evaluation of reference genes and expression of key genes involved in the isoprenoid metabolic pathway of rice leaves after infection by the Southern rice black-streaked dwarf virus

Molecular Biology Reports - The Southern rice black-streaked dwarf virus (SRBSDV), a novel Fijivirus, poses a major threat to rice production in East Asia. Analysis of the gene expression of...     

Silencing by RNAi of the gene for Pns12, a viroplasm matrix protein of Rice dwarf virus, results in strong resistance of transgenic rice plants to the virus

The non-structural protein Pns12 of Rice dwarf virus is one of the early proteins expressed in cultured insect cells, and it is one of 12 proteins that initiate the formation of the viroplasm, the putative site of viral replication. Pns4 is also a non-structural protein, visible as minitubules after nucleation of the viroplasm. We introduced Pns12- and Pns4-specific RNA interference (RNAi) constructs into rice plants. The resultant transgenic plants accumulated short interfering RNAs specific to the constructs. The progeny of rice plants with Pns12-specific RNAi constructs, after self-fertilization, were strongly resistant to viral infection. By contrast, resistance was less apparent in the case of rice plants with Pns4-specific RNAi constructs, and delayed symptoms appeared in some plants of each line. Our results suggest that interference with the expression of a protein that is critical for viral replication, such as the viroplasm matrix protein Pns12, might be a practical and effective way to control viral infection in crop plants.     

Heterologous expression of artificial miRNAs from rice dwarf virus in transgenic rice

In contrast to hairpin RNAs, in which heterogeneous small RNAs are processed from double-stranded RNA to have potential off-target effects on endogenous other genes, artificial miRNAs (amiRNAs) have advantages of exquisite specificity and non-transitivity to thus target individual genes and groups of endogenous genes. Earlier studies showed that amiRNA engineering based on osa-miRNA528 precursor could efficiently trigger endogenous gene silencing and modulate agronomic traits in rice. However, both the expression efficiency of heterologous amiRNAs based on osa-miRNA528 precursor and the correlation of copy number with the relative expression level of amiRNAs remain unknown. In the present study, five amiRNAs (S9-1174, S9-1192, S11-864, S11-868 and S11-869) targeting different sites of S9 and S11 negative strands in rice dwarf virus (RDV) genome were constructed using endogenous osa-miRNA528 precursor as backbone. After identification by Northern blot, two amiRNAs (S9-1174 and S9-1192) targeting S9 negative strand in RDV genome were highly expressed, whereas in three tested amiRNAs targeting S11 negative strand in RDV genome, only two amiRNAs (S11-868 and S11-869) were processed efficiently. T0 generation transgenic rice containing amiRNAs (S9-1174, S9-1192, S11-868 and S11-869) exhibited different expression ratios of amiRNAs, accounting for 90.0, 90.0, 66.7 and 77.8 %, respectively. In addition, combination analysis with the relative amiRNA expression levels and its copy number revealed that the relative expression levels of amiRNAs had no relation to the copy number of T-DNA insert in transgenic rice.     

Hairpin RNA derived from the gene for Pns9, a viroplasm matrix protein of Rice gall dwarf virus, confers strong resistance to virus infection in transgenic rice plants

The nonstructural Pns9 protein of Rice gall dwarf virus (RGDV) accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in host cells infected by viruses in the family Reoviridae. An RNA interference construct was designed to target the gene for Pns9 of RGDV, namely Trigger_G9. The resultant transgenic plants accumulated short interfering RNAs specific for the construct. All progenies from self-fertilized transgenic plants had strong and heritable resistance to RGDV infection and did not allow the propagation of RGDV. By contrast, our transgenic plants remained susceptible to Rice dwarf virus, another phytoreovirus. There were no significant changes in the morphology of our transgenic plants compared with non-inoculated wild-type rice plants, suggesting that genes critical for the growth of rice plants were unaffected. Our results demonstrate that the resistance to RGDV of our transgenic rice plants is not due to resistance to the vector insects but to specific inhibition of RGDV replication and that the designed trigger sequence is functioning normally. Thus, our strategy to target a gene for viroplasm matrix protein should be applicable to plant viruses that belong to the family Reoviridae.     

Disruption of a novel gene for a NAC-domain protein in rice confers resistance to Rice dwarf virus

Rice dwarf virus (RDV) is a serious viral pest that is transmitted to rice plants ( Oryza sativa L.) by leafhoppers and causes a dwarfism in infected plants. To identify host factors involved in the multiplication of RDV, we screened Tos17 insertion mutant lines of rice for mutants with reduced susceptibility to RDV. One mutant, designated rim1-1 , did not show typical disease symptoms upon infection with RDV. The accumulation of RDV capsid proteins was also drastically reduced in inoculated rim1-1 mutant plants. Co-segregation and complementation analyses revealed that the rim1-1 mutation had been caused by insertion of Tos17 in an intron of a novel NAC gene. The rim1-1 mutant remained susceptible to the two other viruses tested, one of which is also transmitted by leafhoppers, suggesting that the multiplication rather than transmission of RDV is specifically impaired in this mutant. We propose that RIM1 functions as a host factor that is required for multiplication of RDV in rice.