P0 protein of cotton leafroll dwarf virus-atypical isolate is a weak RNA silencing suppressor and the avirulence determinant that breaks the cotton Cbd gene-based resistance

Cotton blue disease (CBD) is the most important disease present in cotton crops in South America and cotton leafroll dwarf virus (CLRDV) is the causal agent. The disease has been controlled by sowing cotton varieties resistant to CLRDV. However, in the 2009/10 growing season, an outbreak due to an atypical CLRDV isolate (CLRDV-at) occurred in northwest Argentina. Although CLRDV and CLRDV-at genomes are very closely related, the symptoms they produce in cotton plants are quite different. P0 is the most divergent protein between the isolates and in CLRDV is a silencing suppressor protein. This work characterized the silencing suppressor activity of the P0 protein encoded by CLRDV-at (P0^CL-at) and evaluated its role in Cbd-resistance break in cotton plants. It was demonstrated that P0^CL-at, despite having a mutation in the consensus of the F-box-like motif, was able to suppress local RNA silencing, but displayed lower activity than P0^CL. P0^CL and P0^CL-at showed no differences in the interaction with Gossypium hirsutum SKP1 orthologue (GSK1) and Nicotiana benthamiana SKP1 and both P0 proteins triggered destabilization of ARGONAUTE1. However, when the ability to enhance PVX symptoms was evaluated, P0^CL-at was shown to be a weaker pathogenicity factor than P0^CL in N. benthamiana. Interestingly, trans-expressed P0^CL-at enabled CLRDV to systemically infect CBD-resistant plants, and a chimeric CLRDV-P0^CL-at infectious clone succeeded in establishing infection in CBD-resistant cotton varieties with symptoms resembling those produced by CLRDV-at. These results strongly suggest that P0^CL-at is the avirulence (Avr) determinant involved in breaking cotton Cbd gene-based resistance.     

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.     

Identification of an RNA silencing suppressor encoded by Grapevine leafroll-associated virus 3

GLRaV-3, a member of the Closteroviridae family and type member of the genus Ampelovirus, is involved in the grapevine leafroll disease. Until now no RNA silencing suppressor has been found among viruses of this genus, contrary to what happens with a large number of other viral genera. In the sister genus Closterovirus, RNA silencing suppressors are present in the 3’ end of the genome and have molecular weights close to 20 KDa. To test for RNA suppressing activity screening of p21, p19.6 and p19.7 proteins, coded for in an analogous genomic location of the GLRaV-3 was undertaken. Only p19.7 revealed suppressor activity demonstrated in diverse silencing inducing systems. This suppressor is able to overcome strong silencing inducers and shares several properties with the BYV p21-like family of suppressors of the closteroviruses. This is the first report of an RNA silencing suppressor in the genus Ampelovirus.     

Diverse conserved domains and a positively selected site in the sugarcane streak mosaic virus P1 protein are essential for RNA silencing suppression and protein stability

RNA silencing is one of the conserved antiviral mechanisms in plants, and viruses encode RNA silencing suppressors (RSS) to overcome host RNA silencing and facilitate virus infection. Sugarcane streak mosaic virus (SCSMV; species Sugarcane streak mosaic virus, genus Poacevirus, family Potyviridae) is a major causal agent of sugarcane mosaic disease in many countries in Asia, including China. In this study, we used Agrobacterium co-infiltration to show that the SCSMV P1 protein, rather than the helper component-proteinase (HC-Pro), functions as a strong RSS to suppress local RNA silencing in Nicotiana benthamiana. Mutational analysis indicated that the 15 amino acids (aa; aa 1–15) of the SCSMV P1 N-terminus were not important for RNA silencing suppression, but rather another 15 aa domain (aa 108–122) containing a conserved motif (LFR/KNKQAYIST) was essential for efficient silencing suppression by P1. In addition to the 15 aa (aa 344–358) domain in the P1 N-terminus, another 15 aa domain (aa 65–79) of P1, containing the LXKA motif and one conserved aa (D78), were associated with P1 protein stability. Furthermore, substituting the histidine (H263) residue in P1 with threonine (H263T) or alanine (H263A) also affected P1 protein stability. Notably, the H263 residue is both a positively selected site and part of the serine protease catalytic triad (HDS). Taken together, our data demonstrate that SCSMV P1, and not HC-Pro, plays a functional role in suppressing RNA silencing, and also show that some conserved motifs and a positivelyselected site in the P1 protein are associated with RSS activity and protein stability.     

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.     

HC Pro 3个参与抑制RNA沉默氨基酸位点的鉴定

 马铃薯Y病毒属病毒编码的辅助成分-蛋白酶（helper component-proteinase,HC-Pro）是第一个被鉴定的RNA沉默抑制因子。本研究通过定点突变的方法获得了马铃薯A病毒（Potato virus A,PVA）HC-Pro的3个突变体, 利用农杆菌共浸润的方法分析了这些突变对HC-Pro抑制RNA沉默活性的影响。与野生型HC-Pro处理相比,Phe⁶、Asn¹¹ 缺失突变体的处理中绿色荧光减弱,而Ile²⁵⁰-Gly²⁵¹-Asn²⁵²（IGN）基序中的Ile和Gly分别突变为Asp和Glu的处理中观察不到绿色荧光。该结果表明Phe⁶、Asn¹¹ 和IGN²⁵⁰  3个位点均参与调控HC-Pro的抑制RNA沉默活性。     

四种P450基因调控小菜蛾对氯虫苯甲酰胺的抗性

为研究CYP6家族P450基因在小菜蛾Plutella xylostella代谢氯虫苯甲酰胺中的作用,利用浸叶法测定不同小菜蛾种群3龄幼虫对氯虫苯甲酰的抗性水平,采用实时荧光定量PCR(quantitative realtime PCR,q RT-PCR)方法分析CYP1v3、CYP1v4、CYP6B6和CYP6f这4种P450基因在小菜蛾不同抗性种群体内的表达差异及杀虫剂的短期诱导效应,并通过RNA干扰技术沉默4种P450基因后分析小菜蛾对氯虫苯甲酰胺的敏感性。结果显示,4种P450基因在中等抗性水平小菜蛾种群体内高表达;氯虫苯甲酰胺处理可诱导4种基因显著上调表达。分别沉默4种P450基因后,处理组小菜蛾的P450酶活力显著下降37.60%～54.82%,且处理组小菜蛾的死亡率显著高于对照组;同时沉默4种基因处理组的小菜蛾P450酶活力显著低于其他各处理组。表明4种P450基因可能同时参于小菜蛾对氯虫苯甲酰胺的代谢。     

Possible correlations between the characteristics of Potato leafroll virus isolates occurring in different geographical regions in Tunisia

Biological and molecular characterization supported by transmission efficiency, symptom expression and Open Reading Frame 0 (ORF0) nucleotide sequence analysis were carried out to assess nine isolates of Potato leafroll virus (PLRV) collected from three Tunisian geographic and bioclimatic zones. Plant-to-plant transmission by Tunisian Myzus persicae aphid clones showed high transmission efficiency for all isolates tested. Symptom expression analysis on a Physalis floridana plant test distinguished viral isolates as very severe, severe and mild. The ORF0 sequences of the Tunisian PLRV isolates showed an assignment to two aggregates when compared with GenBank PLRV sequences. A significant correlation between symptom severity and ORF0 nucleotide sequence or between symptom severity and geographic origins of the PLRV isolates was established. However, the transmission efficiency and the ORF0 sequence were not affected by the bioclimatic origin. No significant correlation between transmission and symptom or between transmission and the ORF0 sequence was detected.     

Partial characterization of a non-proteinaceous suppressor of non-specific elicitors from Cladosporium fulvum (syn. Fulvia fulva)

It is our working hypothesis that suppression of the activity of glycoprotein non-specific elicitors (NSE) from fungal cell walls is required in establishment of basic compatibility in the Cladosporium fulvum-tomato system. A suppressor of NSE-induced necrosis on tomato leaves was partially purified from intercellular fluid (IF) obtained from C. fulvum infected, or uninoculated, leaves. The suppressor was stable to treatment with heat, protease, glucosidase, galactosidase, laminarinase, periodate, and mild acid (0·1 N HCl) and base (0·01 N NaOH). Addition of the chelators, EDTA (15 mM) or EGTA (2 mM), to IF resulted in a marked reduction in suppressor activity. Suppressor activity was partially reduced by dialysis of IF, but activity was lost upon dialysis by prior treatment of IF with urea, or with protease which was then inactivated by heating. Activity of a low molecular weight suppressor, partially purified by dialysis and Sephadex G-25 column chromatography, corresponded with a carbohydrate peak. Pectinase or pectinase-generated oligogalacturonides from polypectate suppressed activity of NSE. However, incubation of NSE with pectinase, followed by heat treatment before assay, did not affect NSE activity. It is suggested that low molecular weight suppressor molecules may originate from action of pectolytic enzymes on host cell walls. In addition to these low molecular weight suppressor, native IF, but not heat treated IF, contained proteins that on in vitro incubation with NSE slowly reduced its ability to induce necrosis.     

Comparing p20’s RNA silencing suppressing activity among five phylogenetic groups of <Emphasis Type="Italic">Citrus Tristeza virus</Emphasis>

The p20 protein encoded by the Citrus Tristeza Virus (CTV) was previously identified as a RNA silencing suppressor. In this study, we analyzed the p20’s suppressing activity from five phylogenetic groups of CTV, using the co-infiltration assay of Green fluorescence protein (GFP) gene and the suppressor gene in 16C line Nicotiana benthamiana plants. Green fluorescence, GFP mRNA relative levels and GFP specific siRNAS were compared showing in most cases, only slight differences. Contrary to previous studies, the p20 suppressor was not able to impede neither short range nor systemic spreading of RNA silencing. The suppressor from the phylogenetic group 4 revealed a much reduced activity when compared with the others. At present we still don’t know whether this property is a characteristic of this group or an atypical feature due to a unique point mutation. The differences in the symptom type and intensity originated by isolates belonging to the phylogenetic groups assayed could not be related to differences to the p20 suppressor’s activity.     

RNA silencing against viruses: molecular arms race between Cucumber mosaic virus and its host

Plants have developed RNA silencing as an antiviral defense mechanism. To escape from the plant host’s defenses, viruses have countered their host’s antiviral silencing by producing RNA silencing suppressor proteins (RSSs). Although the mode of action of the majority of viral RSSs has been found to be through double-stranded RNA-binding, viruses have different strategies to counteract the host’s antiviral silencing pathways. The 2b protein of Cucumber mosaic virus, which is one of the most extensively studied viral RSSs, is reviewed here to provide insights on the molecular arms race between viruses and their host plants.     

A new ZTL-type F-box functions as a positive regulator in disease resistance: VIGS analysis in barley against powdery mildew

In our previous studies, we found a new Zeitlupe (ZTL) type F-box protein which is expressed at a higher level upon avirulent pathogen infection (Bozkurt et al., 2007). F-box proteins mark the proteins to be degraded through 26S proteasome system by ubiquitination. Since the information on the role of ubiquitin mediated proteolysis in disease responses is advancing rapidly, we sought to understand the way which F-box functions in resistance response as part of ubiquitin–proteasome pathway. Interestingly, in response to silencing of this F-box gene via BSMV mediated virus induced gene silencing (VIGS) method, barley plants lost resistance towards avirulent pathogen race. The Pallas-01 line having Mla1 R-gene showed hyphae formations when inoculated with avirulent powdery mildew race, Bgh103, after 4-fold silencing. This observation suggests that F-box protein functions as a positive regulator in powdery mildew disease mechanism and broadens the function of ZTL-type F-box proteins, previously known to have roles only in circadian clocks, flowering time control, and phytochrome pathway.     

Fusion proteins of single-chain variable fragments derived from phage display libraries are effective reagents for routine diagnosis of potato leafroll virus infection in potato

ABSTRACT A panel of 11 different single-chain variable fragment antibodies (scFv) that bind to potato leafroll virus (PLRV) has been studied to assess each one's suitability as practical diagnostic tools. The scFv, previously obtained from naive phage display libraries, were expressed in Escherichia coli as fusion proteins. The fusion proteins comprised scFv joined to either the human light chain kappa constant domain (C(L)), an amphipathic helix (Zip), a combination of C(L) and Zip, or alkaline phosphatase (AP/S). The fusion proteins were tested for their ability to detect, or trap on enzymelinked immunosorbent assay (ELISA) plates, PLRV in extracts of infected potato leaves. The tests done with the different scFv fusion proteins were compared with a standard triple-antibody sandwich (TAS)-ELISA that employs a rabbit polyclonal antibody preparation to coat microtiter plates and a monoclonal antibody, SCR3, to detect PLRV. Of 11 scFvC(L) fusion proteins, 7 detected PLRV as readily as SCR3 when used as detecting antibodies in TAS-ELISA. The limit of detection of purified PLRV for the different scFvC(L) fusion proteins ranged from 250 to 5 ng/ml; that for SCR3 is 5 ng/ml. Of the 11 scFv, 4 cross-reacted with some other luteoviruses. Several scFvC(L) and scFvC(L)Zip fusion proteins trapped PLRV from extracts of infected potato leaves as effectively as the polyclonal antibody preparation. Four scFv fusion proteins were used in a stem print assay to detect PLRV, and the results were similar to those obtained in tests using SCR3. The scFvC(L) fusion proteins retained activity for at least 6 months at 4 degrees C, and all scFv fusion proteins were fully active on reconstitution after lyophilization. A fully recombinant ELISA was devised that detected PLRV in extracts of infected potato, with results comparable to those obtained using the standard TAS-ELISA. The advantages of using scFv fusion proteins for the routine detection of plant viruses include the ability to produce large quantities of reagents cheaply in bacterial fermenters and to incorporate them into standardized tests.     

Argonaute1敲除前后尖孢镰刀菌转录组分析比较

 Argonaute蛋白(AGO)介导的沉默复合体在RNA干扰(RNAi)中起着至关重要的作用。为了探究AGO1在尖孢镰刀菌RNAi中的作用机制,本文以粘团专化型尖孢镰刀菌生理1号小种FOX-A8野生型和其AGO1缺失突变体(FOX-A8-△Ago1)的菌丝和孢子为材料,分别进行了RNA提取、Illumina HiSeq 2000高通量转录组测序、差异表达基因(DEGs)的显著富集分析;选择菌丝和孢子中的DEGs 进行实时荧光定量PCR(Quantitative real-time PCR, qRT-PCR)验证。GO通路注释结果显示,相对于野生型菌株,AGO1缺失突变体菌丝中的醇脱氢酶(NADP⁺)、孢子中的CAMK / CAMKL / CHK 1蛋白激酶均显著上调;KEGG通路注释结果显示,相对于野生型菌株,AGO1缺失突变体菌丝中与 MAPK信号通路相关的基因、孢子中与PLD信号通路相关的基因均显著下调;另外,相对于野生型菌株,编码AGO2的基因下调,但是下调不显著。qRT-PCR检测DEGs的表达模式与RNA-Seq分析结果一致,证实了RNA-Seq结果的可靠性。     

Virus transmission by aphids in potato crops

This paper reviews the contribution of vector activity and plant age to virus spread in potato crops. Determining which aphid species are vectors is particularly important for timing haulm destruction to minimize tuber infection by potato virus Y (PVY). Alate aphids of more than 30 species transmit PVY, and aphids such asRhopalosiphum padi, that migrate in large numbers before flights of the more efficient vector,Myzus persicae, appear to be important vectors. Differences in methodology, aphid biotypes and virus strains prevent direct comparisons between estimates of vector efficiencies obtained for aphids in different countries in north western Europe. M. persicae is also the most efficient vector of potato leafroll virus (PLRV), but some clones ofMacrosiphum euphorbiae transmit PLRV efficiently toNicotiana clevelandii and potato test plants. The removal of infected plants early in the season prevents the spread of PLRV in cool regions with limited vector activity. The proportion of aphids acquiring PLRV from infected potato plants decreases with plant age, and healthy potato plants are more resistant to infection later in the season. Severe symptoms of secondary leafroll developed on progeny plants of cv. Maris Piper derived from mother plants inoculated with PLRV in June or July of the previous year. Progeny plants derived from mother plants inoculated in August showed only mild symptoms, but the concentration of PLRV in these plants was as high as that in the plants with severe symptoms.     

Argonaute1敲除前后尖孢镰刀菌转录组分析比较

 Argonaute蛋白(AGO)介导的沉默复合体在RNA干扰(RNAi)中起着至关重要的作用。为了探究AGO1在尖孢镰刀菌RNAi中的作用机制,本文以粘团专化型尖孢镰刀菌生理1号小种FOX-A8野生型和其AGO1缺失突变体(FOX-A8-△Ago1)的菌丝和孢子为材料,分别进行了RNA提取、Illumina HiSeq 2000高通量转录组测序、差异表达基因(DEGs)的显著富集分析;选择菌丝和孢子中的DEGs 进行实时荧光定量PCR(Quantitative real-time PCR, qRT-PCR)验证。GO通路注释结果显示,相对于野生型菌株,AGO1缺失突变体菌丝中的醇脱氢酶(NADP⁺)、孢子中的CAMK / CAMKL / CHK 1蛋白激酶均显著上调;KEGG通路注释结果显示,相对于野生型菌株,AGO1缺失突变体菌丝中与 MAPK信号通路相关的基因、孢子中与PLD信号通路相关的基因均显著下调;另外,相对于野生型菌株,编码AGO2的基因下调,但是下调不显著。qRT-PCR检测DEGs的表达模式与RNA-Seq分析结果一致,证实了RNA-Seq结果的可靠性。     

Induction of defense response of Oryza sativa L. against Pyricularia grisea (Cooke) Sacc. by treating seeds with chitosan and hydrolyzed chitosan

Seeds of rice (Oryza sativa L.) were treated with chitosan and hydrolyzed chitosan at 100, 500 and 1000 mg L⁻¹. After 18 days of germination, spore suspension of Pyricularia grisea was applied. The enzyme activity of phenylalanine ammonia-lyase, β-1-3-glucanase, chitinase and chitosanase in leaves of rice seedlings was evaluated after 24, 72, 120 and 168 h of inoculation. Blast affected area (%) was evaluated 7 and 14 days after spraying spore suspension. Chitosan performance to elicit defense response induction was associated with the concentration and type of chitosan. The activity of most of the enzymes tested was induced in leaves of treated seeds with chitosan and hydrolyzed chitosan at 1000 and 500 mg L⁻¹, respectively. The highest enzyme activities were observed with hydrolyzed chitosan after 72 h however, compared to chitosan, the activity was not maintained during the entire post-inoculation period. The highest control (0 = no lesions) of P. grisea in rice seedlings was observed at 1000 mg L⁻¹ in both chitosan and hydrolyzed chitosan treated leaves. Symptoms of infection by P. grisea were evident after 14 days evaluation date, but according to the standard scale proposed by the International Rice Research Institute, these symptoms fell into the resistance category of blast diseases.