The DNA damage signal transducer ortholog <Emphasis Type="Italic">Mop53BP1</Emphasis> is required for proper appressorium differentiation and pathogenicity in <Emphasis Type="Italic">Pyricularia oryzae</Emphasis>

Appressorium differentiation, one of the most important steps in pathogenesis by the rice blast fungus, Pyricularia oryzae, is strongly coordinated with the cell cycle. In this study, we identified an ortholog gene of 53BP1, which encodes a signal transducer protein that participates in G2-M cell cycle checkpoint in higher eukaryotes, in the genome of P. oryzae and characterized the phenotype of deletion and overexpression mutants. Deletion mutants showed no significant deficiency in vegetative growth compared to wild-type and complemented strains, even on the media containing DNA-damaging agents. However, these null mutants had abnormal appressoria and formed more appressoria per conidium than in the wild type and were unable to penetrate the epidermis of rice leaves. eGFP-fused Mop53BP1 and qRT-PCR analyses revealed that Mop53BP1 is expressed during the first hours of appressorium formation. In addition, in overexpression mutants, Mop53BP1 localized to nuclei during all stages of appressorium maturation and penetration, and the mutants were resistant to the microtubule inhibitor benomyl, suggesting that Mop53BP1 is nuclear protein and may have some role related to microtubules.     

EDS1-mediated basal defense and SA-signaling contribute to post-invasion resistance against tobacco powdery mildew in Arabidopsis

Arabidopsis-powdery mildew is a unique pathosystem in dissecting the molecular mechanism of plant-biotrophic fungus interactions. To increase the diversity of powdery mildew pathogens, we identified a tobacco powdery mildew, designated Gc SICAU1, which belonged to Golovinomyces cichoracearum (Gc) based on the internal transcribed spacer (ITS) rDNA fragment sequences. Inoculation test in laboratory conditions revealed that Gc SICAU1 could colonize on wild type Arabidopsis Col-0 leaves, but failed to grow as abundantly on tobacco leaves, indicating that Gc SICAU1 has completely overcome the pre-invasion resistance and its colonization is hindered by the post-invasion resistance in Arabidopsis. The growth of Gc SICAU1 in the fls2, efr-1 and pen1-1 mutants displayed no obvious difference from that of wild type plants. In contrast, salicylic acid (SA)-signaling deficient mutants such as pad4-1 and eds5-1, but not ethylene-signaling mutants, exhibited increased susceptibility to Gc SICAU1. Moreover, eds1-2 and pad4-1/sid2-1 were fully susceptible to Gc SICAU1 and the disease symptom was comparable to Gc SICAU1 on tobacco leaves. These data indicate that EDS1-mediated basal defense and SA-signaling play critical roles for post-invasion resistance against this pathogen. Taken together, our data demonstrated that Gc SICAU1 is a pathogen weakly virulent to Arabidopsis and can be used as a tool to finely dissect post-invasion resistance mechanisms.     

Antiproliferative effect of azadirachtin A on Spodoptera litura Sl-1 cell line through cell cycle arrest and apoptosis induced by up-regulation of p53

In order to illustrate the mechanism of cell cycle arrest and apoptosis induced by botanical insecticide, the effects of cell proliferation inhibition and apoptosis induction by azadirachtin A were investigated in Spodoptera litura cell line Sl-1 in vitro. The cell proliferation, cell cycle division, mitotic and apoptotic figure of nucleus, and the expression of related proteins were detected by a variety of methods, including MTT assay, flow cytometry, fluorescence microscope observation, and Western blot technique. Sl-1 cells grew in a dose-dependent inhibition manner and the proliferation inhibition rates were 18.95%, 33.45% and 41.00%, after 48 h of treatment with azadirachtin A at the concentrations of 0.1, 0.5 and 1.0 μg/mL, respectively. Sl-1 cell cycle was arrested in G2/M phase with lower expression of cyclin B1. The apoptosis rates were found to be 19.71%, 23.40% and 24.91% after 48 h of treatment with 0.1, 0.5, and 1.0 μg/mL of azadirachtin A, which increased with the activities of caspase-3. The up-regulation of p53 was observed before apoptosis and cell cycle arrest occurred. These findings suggest that p53 protein was involved in cell cycle arrest, apoptosis induction and cell proliferation inhibition when the insect cultured cells were treated with azadirachtin A.     

Harpin-elicited hypersensitive cell death and pathogen resistance require the NDR1 and EDS1 genes

Plants sprayed with harpin, a bacterial protein that induces hypersensitive cell death (HCD), develop systemic acquired resistance (SAR) without macroscopic necrosis. HCD sometimes accompanies the development of resistance conferred by resistance (R) genes. In Arabidopsis, some R genes require one or both of the signalling components NDR1 and EDS1 for function. This study addresses whether HCD, NDR1 and EDS1 are required for induction of SAR by harpin. When Arabidopsis and tobacco leaves were sprayed with harpin, microscopic hypersensitive response (micro-HR) lesions developed. Systemic expression of PR genes and the development of resistance were accompanied by micro-HR, except in the ndr1-1 mutant, in which harpin induced micro-HR without the development of resistance or expression of the PR-1 gene. Cell death and resistance did not occur following treatment with harpin in plants that could not accumulate salicylic acid. Harpin also failed to induce resistance in Arabidopsis eds1-1 mutants. Therefore, harpin-induced resistance seems to develop concomitantly with cell death and resistance requires NDR1 and EDS1.     

SGT1 contributes to maintaining protein levels of MEK2DD to facilitate hypersensitive response-like cell death in Nicotiana benthamiana

Gene silencing revealed that the mitogen-activated protein kinase (MAPK) cascade in Solanaceae consisted with MEK2-WIPK/SIPK, is required for R protein-induced hypersensitive response (HR) cell death and/or resistance. Overexpression of MEK2^DD results in HR-like cell death. MEK2^DD is a phospho-mimic and constitutive active form harboring mutations at putative phosphorylation sites of upstream MAPKKK. The molecular mechanism that induces HR-like cell death is unknown. Here we report SGT1 is required for the accumulation of MEK2^DD protein, not MEK2^WT. Virus-induced gene silencing of SGT1 resulted in low protein accumulation of MEK2^DD. This result suggests that SGT1 has a positive role in the accumulation of the MEK2 active form protein to facilitate signal transduction.     

The carboxyterminal processing protease of D1 protein: Herbicidal activity of novel inhibitors of the recombinant and native spinach enzymes

The carboxyterminal processing protease of D1 protein (CtpA) is predicted to be an excellent target for the discovery of a general broad-spectrum herbicide. Directed and random screening of compounds against recombinant spinach CtpA (rCtpA) has led to the discovery of five different chemical classes of inhibitors. Lead compounds from each inhibitor class were investigated for their in vitro effects on the activities of both recombinant and native spinach CtpA. All of the lead compounds have K_i values of less than 50 μM when tested against rCtpA, and all except one showed competitive inhibition. Results from partially purified native CtpA from spinach were similar to those from the recombinant form of the enzyme, thus validating the use of rCtpA in the inhibitor screen. Compounds from three of the classes of CtpA inhibitors show in vivo herbicidal activity against Arabidopsis thaliana when applied either by addition to growth media or by spraying the leaves. Transgenic Arabidopsis plants which over-express CtpA showed greater resistance to the compounds than wild-type plants providing evidence that these inhibitors are directly acting against CtpA.     

Transgenic expression of a sorghum gene (SbLRR2) encoding a simple extracellular leucine-rich protein enhances resistance against necrotrophic pathogens in Arabidopsis

Plant leucine-rich repeat (LRR) domain-containing proteins are known to play important roles in signaling transduction and defense responses. In sorghum, SbLRR2 is pathogen-inducible gene encoding a simple extracellular LRR protein. Here, we demonstrated an earlier and stronger expression of SbLRR2 in a sorghum resistant genotype in comparison to a susceptible genotype following inoculation with the anthracnose pathogen (Colletotrichum sublineolum). In addition, SbLRR2 expression was found to be induced strongly by methyl-jasmonate treatment. Functional analysis was performed in SbLRR2 over-expression (OE) Arabidopsis plants, which showed enhanced resistance against the necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola. In addition, the OE lines were found to have elevated expression of several jasmonate acid (JA)-associated genes and higher endogenous JA contents. Hence, the SbLRR2-mediated defense responses in transgenic Arabidopsis are likely to be dependent on JA-signaling through increased JA production. On the other hand, the OE lines remained susceptible to Pseudomonas syringae pv. tomato like the wild type plants. Consistently, there was no up-regulation of salicylic acid (SA) defense marker gene expression or SA levels in the OE lines. Our results suggested that SbLRR2 is potentially useful for enhancing resistance against necrotrophic pathogens in transgenic dicot crops.     

Cry1Ac的绿色荧光蛋白GFP标记及其在棉铃虫幼虫中肠的定位分布

为了明确Cry1Ac蛋白在棉铃虫体内与中肠组织的相互作用,采用重叠PCR方法将Bt-cry1Ac基因和绿色荧光蛋白GFP基因融合,构建含Cry1Ac毒蛋白和绿色荧光蛋白GFP原核表达载体,并在大肠杆菌大量表达。利用荧光显微镜观察发现,表达Cry1Ac-GFP融合蛋白的大肠杆菌在蓝光激发下发出绿色荧光。将含有融合蛋白的菌液拌入人工饲料饲喂3龄棉铃虫幼虫96h,取棉铃虫幼虫中肠做冰冻切片并在荧光显微镜下观察。结果显示,取食含有Cry1Ac-GFP融合蛋白饲料的棉铃虫幼虫中肠能够发出强烈荧光。比较Cry1Ac杀虫蛋白敏感和抗性棉铃虫幼虫中肠的发光部位,敏感棉铃虫幼虫的中肠围食膜已经消失,肠壁细胞发出强烈的荧光,而抗性棉铃虫的围食膜较健全并发出荧光。     

Herbicidal cyanoacrylates with antimicrotubule mechanism of action

The herbicidal mode of action of the new synthetic cyanoacrylates ethyl (2Z)-3-amino-2-cyano-4-ethylhex-2-enoate (CA1) and its isopropyl ester derivative CA2 was investigated. For initial characterization, a series of bioassays was used indicating a mode of action similar to that of mitotic disrupter herbicides such as the dinitroaniline pendimethalin. Cytochemical fluorescence studies including monoclonal antibodies against polymerized and depolymerized tubulin and a cellulose-binding domain of a bacterial cellulase conjugated to a fluorescent dye were applied to elucidate effects on cell division processes including mitosis and microtubule and cell wall formation in maize roots. When seedlings were root treated with 10 microM of CA1 or CA2, cell division activity in meristematic root tip cells decreased within 4 h. The chromosomes proceeded to a condensed state of prometaphase, but were unable to progress further in the mitotic cycle. The compounds caused a complete loss of microtubular structures, including preprophase, spindle, phragmoplast and cortical microtubules. Concomitantly, in the cytoplasm, an increase in labelling of free tubulin was observed. This suggests that the herbicides disrupt polymerization and microtubule stability, whereas tubulin synthesis or degradation appeared not to be affected. In addition, cellulose labelling in cell walls of root tip cells was not influenced. The effects of CA1 and CA2 were comparable with those caused by pendimethalin. In transgenic Arabidopsis plants expressing a green fluorescent protein-microtubule-associated protein4 fusion protein, labelled arrays of cortical microtubules in living epidermal cells of hypocotyls collapsed within 160 min after exposure to 10 microM CA1 or pendimethalin. Moreover, a dinitroaniline-resistant biotype of goosegrass (Eleusine indica (L) Gaertn) with a point mutation in alpha-tubulin showed cross-resistance against CA1 and CA2. The results strongly indicate that the cyanoacrylates are a new chemical class of herbicide which possess the same antimicrotubule mechanism of action as dinitroanilines, probably including interaction with the same binding site in alpha-tubulin.     

Turnip crinkle virus with nonviral gene cancels the effect of silencing suppressors of P19 and 2b in Arabidopsis thaliana

In plants, green fluorescent protein (GFP) has become a preferred molecular marker for gene expression and cellular localization, and plant viral vectors are valuable tools for heterologous gene expression. Some plant viruses have been used for expression of GFP, and the activities of these viruses are barely affected by the extra GFP gene. In contrast, the packaging and the length of Turnip crinkle virus (TCV) genome is strictly limited when foreign genes are inserted into the coding sequences of TCV genome. In this report, we removed the silencing suppressor p38 from TCV, and constructed GFP derivatives of TCV. Then the resulting TCV mutants were used to infect Arabidopsis plants containing mutations in key silencing pathway genes, including triple dcl2/dcl3/dcl4, dcl2, dcl4 and ago mutant plants. Our results demonstrate that the activity of TCV is affected by nonviral GFP insert in Arabidopsis plants, and RNA silencing appears not play an important role. AGOs appear to be more efficient at slicing RNAs of viral origin, especially AGO2 and AGO7. Although the viral suppressors of RNA silencing (VSRs) P19 and 2b can enhance the accumulation of viral RNAs, neither P19 nor 2b can significantly increase the expression of TCV mutants with nonviral genes. TCV is an example of an RNA virus that is recalcitrant to add nonviral gene sequences.     

The herbicide flamprop-M-methyl has a new antimicrotubule mechanism of action

BACKGROUND: The herbicidal mode of action of flamprop‐M‐methyl [methyl N‐benzoyl‐N‐(3‐chloro‐4‐fluorophenyl)‐D ‐alaninate] was investigated. RESULTS: For initial characterization, a series of bioassays was used, which indicated a mode of action similar to that of mitotic disrupter herbicides. Cytochemical fluorescence studies, which included monoclonal antibodies against polymerized tubulin, were applied to elucidate effects on mitosis and microtubule assembly in maize roots. When seedlings were root treated with 50 µM of flamprop‐M‐methyl, cell division activity in meristematic root tip cells ceased within 4 h. The compound severely disturbed the orientation of spindle and phragmoblast microtubules, leading to defective spindle and phragmoblast structures. Cortical microtubules were only slightly affected. In late anaphase and early telophase cells, phragmoblast microtubules were disorganized in multiple arrays that hampered regular cell plate deposition in cytokinesis. Microtubules of the spindle apparatus were found attached to chromosomal kinetochores, but did not show regular organization associated with a zone of microtubule‐organizing centres at the opposite ends of the cell. On account of this loss of spindle organization, chromosomes remained in a condensed state of prometaphase or metaphase. Unlike known microtubule disrupter herbicides, flamprop‐M‐methyl and its biologically active metabolite flamprop did not inhibit soybean tubulin polymerization to microtubules in vitro at 50 µM . In contrast, soybean plants responded sensitively to the compounds. CONCLUSION: The results indicate that flamprop‐M‐methyl is a mitotic disrupter herbicide with a new antimicrotubule mechanism of action that affects orientation of spindle and phragmoblast microtubules, possibly by minus‐end microtubule disassembly. Copyright © 2008 Society of Chemical Industry     

Isolation,production and in vitro effects of the major secondary metabolite produced by Trichoderma species used for the control of grapevine trunk diseases

Antibiosis has been shown to be an important mode of action by Trichoderma species used in the protection of grapevine pruning wounds from infection by trunk pathogens. The major active compound from Trichoderma isolates known to protect grapevine pruning wounds from trunk pathogen infection was isolated and identified. The compound, a 6‐pentyl‐α‐pyrone (6PP), was found to be the major secondary metabolite, by quantity, which accumulated in the culture filtrate of T. harzianum isolate T77 and the two T. atroviride isolates UST1 and UST2. Benzimidazole resistant mutants generated from these isolates also produced 6PP as their main secondary metabolite, except for a mutant of T77 that had lost its ability to produce 6PP. The isolates UST1 and UST2 were co‐cultured with the grapevine trunk pathogens Eutypa lata and Neofusicoccum parvum in a minimal defined medium and a grapevine cane‐based medium (GCBM). Co‐culturing UST1 with N. parvum induced 6PP production in the minimal defined medium and the GCBM. The production of 6PP by UST2 was induced in the GCBM, while co‐culturing with the two trunk pathogens either reduced or had no effect on 6PP production. Mycelial growth and ascospore/conidia germination of E. lata, N. australe, N. parvum and Phaeomoniella chlamydospora were inhibited by 6PP in a concentration‐dependent manner. The results show that the presence of N. parvum and grapevine wood elicits the production of 6PP, suggesting that this metabolite is involved in Trichoderma–pathogen interactions on grapevine pruning wounds.     

Race-specific elicitors from the Peronospora parasitica / Arabidopsis thaliana pathosystem

Intercellular washing fluids (IWFs) were isolated from compatible interactions ofPeronospora parasitica with Arabidopsis. IWFs were filter-sterilized then infiltrated into leaves of resistant and susceptible Arabidopsis genotypes. P. parasitica isolates WELA (virulent on Landsberg erecta (La-er) and Weiningen (Wei-0) but avirulent on Columbia (Col-0) and RLD) and NOCO (virulent on Col-0 and RLD but avirulent on La-er) were used. A “reciprocal check” pattern of necrosis-induction was observed in which IWFs from a compatible interaction caused necrosis in leaves of the Arabidopsis genotypes resistant to the “parent” fungal isolate but not in those genotypes susceptible to it. Analysis of variance showed that the interaction between fungal isolate and necrosis in host genotypes was highly significant. The necrosis-inducing activity was significantly correlated with a high degree of asexual sporulation in the compatible interaction from which the IWF was isolated. However, the necrosis-inducing activity was not correlated with the protein content of the IWF.