Generation and evaluation of movement protein‐specific single‐chain antibodies for delaying symptoms of Tomato spotted wilt virus infection in tobacco

This study investigated whether single‐chain antibodies (scFvs) specific for a viral movement protein could accumulate in the plant cell cytosol and restrict viral systemic infection in plants. Nine chicken scFv fragments against the Tomato spotted wilt virus (TSWV) movement protein (NS_M) were isolated by phage display. Soluble scFvs were produced in bacteria and the NS_M binding activity of purified scFvs was confirmed. The nine scFv genes were cloned into a plant expression vector enabling recombinant protein accumulation in the plant cell cytosol. Immunoblot analysis demonstrated that two of the nine chicken scFvs accumulated to high levels (5·9 and 8·0% of total soluble protein). Bioassays of viral infection using transgenic tobacco plants producing NS_M‐specific chicken scFvs showed delayed symptom development when compared to non‐transgenic control plants, indicating that expression of antibodies recognizing the TSWV movement protein is a potential strategy for generating resistant plants.     

Non-structural plum pox potyvirus proteins detected by immunogold labelling

Plum pox potyvirus (PPV) induces in infected Nicotiana clevelandii cells characteristic crystalline inclusions known as nuclear inclusions (NI) when located in the nucleus and as dense material (Dm) when located in the cytoplasm. Crystalline inclusions contain protease (NIa) and RNA-dependent RNA polymerase (NIb) proteins. It is now well established for all potyviruses that cylindrical inclusions contain CI helicase ATPase protein (Martin et al., 1992). The intracellular location of other non-structural PPV proteins remains unknown. Using Escherichia coli expression vectors, specific antibodies were obtained against P1, P3, 6K2 and NIb PPV proteins for which antibodies were not yet available. As expected, NIb antiserum labelled crystalline inclusions. P1, P3 and 6K2 proteins were present in both types of crystalline inclusions found in the nucleus and in the cytoplasm of PPV-infected leaves of N. clevelandii, suggesting that nuclear inclusions and dense material were composed of the same proteins. This composition is discussed.     

Production of plants of Nicotiana benthamiana and Prunus domestica transgenic for plum pox potyvirus coat protein,and demonstration of PPV resistance in transformed N. benthamiana1

Leaf discs of Nicotiana benthamiana plants were transformed with Agrobacterium tumefaciens and transgenic plants expressing plum pox potyvirus (PPV) coat protein (CP) were generated. Homozygous R₂ progeny from these plants were inoculated with PPV. Plants were scored for the appearance of symptoms and tested for infection by DAS-ELISA. Various levels of resistance were obtained after an initial stage in which PPV was able to multiply in all the transgenic plants. Within 2–3 weeks after inoculation, the transgenic resistant plants fully recovered from virus infection. Conversely, control and susceptible transgenic lines developed severe symptoms and high virus titres. Prunus domestica (plum) was transformed by inoculating hypocotyl slices with A. tumefaciens containing a binary plasmid which included the NPTII, GUS, and PPV CP genes within its T-DNA region. Transgenic shoots were rooted and established in the glasshouse. Analysis of selected transformants by PCR showed that the engineered foreign genes had been integrated, including that for PPV CP. Histological assays on young leaves of these putative transformants gave a positive reaction. This suggests that all genes transferred are expressed in these transformed plums.     

Viral infection‐induced endoplasmic reticulum stress and a membrane‐associated transcription factor NbNAC089 are involved in resistance to virus in Nicotiana benthamiana

Endoplasmic reticulum (ER) stress may induce two cell defence pathways, the unfolded protein response (UPR) or programmed cell death (PCD) upon unmitigated stress. This study confirmed that viral infection could induce ER stress through changing ER morphology and up‐regulating ER stress‐related genes, including NbNAC089. AtNAC089 serves as an ER stress sensor to regulate PCD in Arabidopsis. In this study, Nicotiana benthamiana NbNAC089 was identified. The gene encoded a 409 amino acid protein with a putative transmembrane domain near the C‐terminus and a NAC domain at the N‐terminus. NbNAC089 was localized to the ER membranes, and a truncated form of NbNAC089, lacking the transmembrane domain, was localized to the nucleus. Meanwhile, the full length of NbNAC089 was activated and cleaved in response to viral infection. The results suggest that the native protein may be translocated to the nucleus by release from the membrane during viral infection. Knock‐down of NbNAC089 in N. benthamiana increased susceptibility to Tobacco mosaic virus or Cucumber mosaic virus, and, in addition, promoted up‐regulation of UPR genes but impaired up‐regulation of PCD genes. These results show that NbNAC089 is a negative regulator of UPR and a positive regulator of PCD, and plays a role in the process of viral infection.     

酵母pac1基因介导对葡萄B病毒的抗性

为明确广谱性抗病毒基因—酵母pac1基因对葡萄B病毒(Grapevine virus B,GVB)的抗性效果,通过农杆菌介导的遗传转化,将pac1基因导入西方烟37B,对转基因植株进行PCR鉴定及Southern blot分析,通过病毒摩擦接种观察症状以及实时荧光定量RT-PCR检测植株体内病毒含量,并对转基因植株抗病性进行初步鉴定。结果表明,目的基因pac1成功导入并整合至西方烟37B基因组,共获得10个转基因株系。不同株系的T1代烟草中阳性植株比例为16.7%~72.7%,表明目的基因可成功遗传到子代。接种病毒后转基因植株普遍延迟发病,但后期症状与非转基因对照相似,其中仅1个转基因株系B6具有不表现典型症状等抗性反应。接种植株病毒含量检测中,所有转基因植株均检测到病毒存在,但表现为抗病的B6株系中病毒含量显著低于非转基因对照,表明该转基因植株虽不能完全抵抗GVB侵染,但对GVB具有耐病性。     

Increased resistance to fusarium wilt in transgenic tobacco lines co‐expressing chitinase and wasabi defensin genes

Marker‐free transgenic tobacco (Nicotiana tabacum) lines containing a chitinase (ChiC) gene isolated from Streptomyces griseus strain HUT 6037 were produced by Agrobacterium‐mediated transformation. One marker‐free transgenic line, TC‐1, was retransformed with the wasabi defensin (WD) gene, isolated from Wasabia japonica. Of the retransformed shoots, 37% co‐expressed the ChiC/WD genes, as confirmed by western and northern analyses. Southern blot analysis showed that no chromosomal rearrangement was introduced between the first and the second transformation. Transgenic lines either expressing ChiC or WD, or co‐expressing both genes were challenged with Fusarium oxysporum f.sp. nicotianae (Fon). Assessment of in vitro plant survival in the presence of Fon showed that transgenic lines co‐expressing both genes had significantly enhanced protection against the fungus (infection indices 0·0–1.·2) compared with corresponding isogenic lines expressing either of the genes (infection indices 2·5–9·8). Whole‐plant infection indices in transgenic lines were significantly related (r = 0·93, P < 0·01) to the extent of root colonization of the host, which ranged from 2·1% to 11·3% in lines co‐expressing both genes, and from 16·8% to 37·7% in lines expressing just one of the genes (compared with 86·4% in non‐transformed controls). Leaf extracts of transgenic lines also inhibited mycelial growth of Fon in vitro and caused hyphal abnormalities.     

马铃薯Y病毒复制酶基因不同位置cDNA区段介导对PVY的不同抗性

为探究靶序列位置对RNA介导的病毒抗性产生的影响,利用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增马铃薯Y病毒(Potato virus Y,PVY)复制酶基因(nuclear inclusion b,NIb)不同位置的cDNA区段,反向插入双元载体pROKII中,构建了发夹RNA(hairpin RNA,hpR-NA)结构的植物表达载体。将构建的植物表达载体采用冻融法转入农杆菌LBA4404,叶盘法转化烟草NC89,获得转基因植株。攻毒试验表明:PVYNIb基因不同位置cDNA区段介导的对PVY的抗性存在显著差异;3′端1/2处和中间位置的序列可介导高水平的病毒抗性,抗性植株的比例在50%以上,而5′端、5′端1/2处和3′端的序列介导的抗性效率较低,抗性植株的比例仅为10%~30%。Northern杂交显示:抗病植株中RNA的积累量明显低于同类型的感病植株,抗性与RNA积累量呈负相关;抗病转基因植株中有siRNA存在,表明病毒抗性是由RNA介导的。     

Evaluation of the resistance of transgenic potato plants expressing various levels of Cry3A against the Colorado potato beetle (Leptinotarsa decemlineata Say) in the laboratory and field

BACKGROUND: The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say, is a destructive pest. The CPB is a quarantine pest in China, but has now invaded the Xinjiang Uygur Autonomous Region and is continuing to spread eastwards. To control the damage and overspreading, transgenic potato plants expressing Cry3A toxin were developed, and their resistance to CPB was evaluated by bioassays in the laboratory and field in 2009, 2010 and 2011. RESULTS: The insect resistance of the high‐dose (HD) transgenic lines was significantly greater than the middle‐dose (MD) and low‐dose (LD) transgenic lines regarding leaf consumption, biomass accumulation and mortality. The HD and MD transgenic lines showed 100% mortality when inoculated with first‐ and second‐instar larvae; however, the LD transgenic lines showed about 50% mortality. The HD transgenic lines exhibited a significantly higher yield than the MD and LD transgenic lines owing to their high CPB resistance. CONCLUSION: Commercially available transgenic potato plants with above 0.1% Cry3A of total soluble protein and NT control refugia could control damage, delay adaptation and halt dispersion eastwards. The two HD transgenic lines developed in this study, PAH1 and PAH2, are ideal for use as cultivars or germplasm to breed new cultivars. Copyright © 2012 Society of Chemical Industry     

Induction of systemic resistance against Cucumber mosaic virus by Penicillium simplicissimum GP17‐2 in Arabidopsis and tobacco

The plant growth‐promoting fungus, Penicillium simplicissimum GP17‐2, was evaluated for its ability to induce resistance against Cucumber mosaic virus (CMV) in Arabidopsis thaliana and tobacco plants. Treatment with barley grain inoculum (BGI) of GP17‐2 significantly enhanced fresh weight, dry weight and leaf number of A. thaliana and tobacco plants 6 weeks after planting. Two weeks after CMV inoculation, all plants treated with BGI of GP17‐2 or its culture filtrate (CF) showed a significant reduction in disease severity compared with non‐treated control plants, which exhibited severe mosaic symptoms by the end of the experiment. The enzyme‐linked immunosorbent assay (ELISA) demonstrated that CMV accumulation was significantly reduced in plants treated with GP17‐2 or its CF relative to control plants. Based on RT‐PCR, plants treated with GP17‐2 (BGI or CF) also exhibited increased expression of regulatory and defence genes involved in the SA and JA/ET signalling pathways. These results suggested that multiple defence pathways in A. thaliana and tobacco were involved in GP17‐2‐mediated resistance to CMV, although neither the transgenic NahG line, nor the npr1, jar1 or ein3 mutants disrupted the response in A. thaliana. This is the first report to demonstrate the induction of systemic resistance against CMV by GP17‐2 or its CF.     

Host‐induced gene silencing in the necrotrophic fungal pathogen Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic fungus that causes a devastating disease called white mould, infecting more than 450 plant species worldwide. Control of this disease with fungicides is limited, so host plant resistance is the preferred alternative for disease management. However, due to the nature of the disease, breeding programmes have had limited success. A potential alternative to developing necrotrophic fungal resistance is the use of host‐induced gene silencing (HIGS) methods, which involves host expression of dsRNA‐generating constructs directed against genes in the pathogen. In this study, the target gene chosen was chitin synthase (chs), which commands the synthesis of chitin, the polysaccharide that is a crucial structural component of the cell walls of many fungi. Tobacco plants were transformed with an interfering intron‐containing hairpin RNA construct for silencing the fungal chs gene. Seventy‐two hours after inoculation, five transgenic lines showed a reduction in disease severity ranging from 55·5 to 86·7% compared with the non‐transgenic lines. The lesion area did not show extensive progress over this time (up to 120 h). Disease resistance and silencing of the fungal chs gene was positively correlated with the presence of detectable siRNA in the transgenic lines. It was demonstrated that expression of endogenous genes from the very aggressive necrotrophic fungus S. sclerotiorum could be prevented by host induced silencing. HIGS of the fungal chitin synthase gene can generate white mould‐tolerant plants. From a biotechnological perspective, these results open new prospects for the development of transgenic plants resistant to necrotrophic fungal pathogens.     

利用RNA介导的抗病性获得高度抗马铃薯Y病毒的转基因烟草

 以马铃薯Y病毒坏死株系(PVY^N)的RNA为模板,应用反转录-聚合酶链式反应(RT-PCR)方法,扩增出长度为801 bp的非翻译的马铃薯Y病毒外壳蛋白基因。将扩增的片段克隆到pBSK的BamHI和KpnI之间并进行了序列测定。用BamHI和KpnI从重组克隆载体上切下该基因并插入到质粒pROKII内得到植物表达载体pPVYCP。通过根癌农杆菌(LBA4404)介导的方法转化烟草NC89,经卡那霉素抗性筛选、PCR和Southern blot检测,获得82株转基因植株。Northern blot和Western blot分析表明,转基因植株只在RNA水平上得到了表达。抗病性试验表明转基因植株之间抗性水平存在着差异,其中有7株是对PVY^N高度抗病性的植株。转基因植株抗病特异性试验初步表明,对PVY^N表现高度抗病的植株对PVY^O也具有高度抗病性。     

Significance of the heterologous encapsidation of zucchini yellow mosaic potyvirus in transgenic plants expressing plum pox potyvirus capsid protein1

Transgenic Nicotiana benthamiana plants expressing the coat protein of an aphid-transmissible strain of plum pox potyvirus (PPV-D) were infected with an aphid non-transmissible strain of another potyvirus, zucchini yellow mosaic potyvirus (ZYMV-NAT). Non-viruliferous Myzus persicae could acquire and transmit ZYMV-NAT from these plants but not from infected N. benthamiana control plants (not transformed, or transformed by the vector alone). Immunosorbent electron microscopy experiments using the decoration technique revealed that ZYMV-NAT virus particles in the infected transgenic plants expressing the PPV coat protein could be coated not only with ZYMV antibodies but also, on segments of the particles, with PPV antibodies. This suggests that aphid transmission of ZYMV-NAT occurred through heterologous encapsidation, and reveals a potential risk of releasing genetically engineered plants expressing viral coat proteins into the environment.     

A novel pair of universal primers for the detection of potyviruses

A novel pair of universal primers was developed to detect potyvirus species after conserved sites were identified using all full‐length potyvirus sequences available by 2005. The breadth of specificity of the new primers, NIb2F and NIb3R, was investigated and compared with the specificity of two routinely used primer pairs in plant virus diagnostic laboratories. RNA from 40 potyvirus isolates representing 23 recognized and three possible new species was tested. Reactions with NIb2F and NIb3R produced amplicons of 350 bp from all 40 virus isolates tested. Reactions with the previously published WCIEN and Potyvirid primers amplified cDNA from 32 and 21 isolates, representing possibly 21 and 15 species, respectively. The identity of 12 unknown potyvirus isolates was confirmed by sequencing and three were found to be potentially distinct potyvirus species. Gel banding patterns from reactions with NIb2F and NIb3R were simpler to interpret than those from reactions with the other two primer sets; fewer products were visible and the cDNA fragments were less variable in size. RT‐PCR with the novel primers is predicted to be able to detect virus isolates from all major groups within the genus Potyvirus and its reliability makes it well suited for use as a routine diagnostic assay.     

The glutamate dehydrogenase gene gdhA increased the resistance of tobacco to glufosinate

The gene gdhA from Escherichia coli, that encodes a NADPH‐dependent glutamate dehydrogenase (GDH), directs a novel pathway in transgenic plants that allows an increase in ammonium assimilation. Glufosinate leads to plant death by the irreversible inhibition of glutamate synthetase (GS) leading to a disruption of subsequent GS‐related processes resulting in elevated ammonium and disruption of photorespiration. Therefore, it was speculated that the gdhA‐transformed plants may exhibit a novel mechanism of resistance to glufosinate by altered activity of the GDH‐directed pathway(s) and subsequently related processes. Studies were conducted in the greenhouse to evaluate the resistance of tobacco plants containing the gdhA gene to glufosinate. Five tobacco genotype lines were investigated including a non‐transformed control line, a positive control line and three transformed lines with levels of increasing GDH activity directed by the gdhA gene. Plants transformed with the gdhA gene expressed up to six times increased level of resistance (GR₅₀) to glufosinate compared with the non‐transformed control, which is 100 times less resistant than plants transformed with the bar gene. The GDH activity among lines was highly correlated (r² = 0.9903) with the level of herbicide resistance. Thus, the use of the E. coli gdhA gene in plant transformations can provide an additional mechanism for resistance to glufosinate.     

内质网应激因子NAC089突变体的创建及其对病毒侵染胁迫的响应

 有研究表明烟草花叶病毒(Tobacco mosaic virus, TMV)或黄瓜花叶病毒(Cucumber mosaic virus, CMV)侵染烟草能够激活转录因子NbNAC089,从ER膜移至细胞核。为进一步阐释内质网应激因子NbNAC089对病毒侵染胁迫的响应机制,利用CRISPR/Cas9技术构建敲除载体,经烟草遗传转染获得NbNAC089基因突变植株。植株接种病毒后采用qRT-PCR检测病毒CP基因和寄主UPR基因的表达。结果表明:CRISPR/Cas9系统定点敲除NbNAC089基因后,目的基因靶位点序列有碱基的置换与缺失。正常生长条件下,转基因植株与野生型无差异。植株接种TMV-GFP后24~96 h,突变体中UPR基因(BiP、bZIP28、bZIP60)的表达量显著高于野生型;接种TMV-GFP后2~6 d突变体中病毒的积累量和扩展速度显著高于野生型。表明NbNAC089为UPR的抑制因子,对病毒增殖具有负调控作用。     

Complementation of coat protein mutants of pepper huasteco geminivirus in transgenic tobacco plants

ABSTRACT The role of the pepper huasteco virus (PHV) coat protein (CP) gene during the infection was investigated in three different hosts by using mutations that produced truncated proteins and by complementation assays in transgenic plants. The infectivity analysis revealed that mutants that express truncated CP (CP7 and CP191) behave like the wild-type virus when inoculated onto pepper and Nicotiana benthamiana plants in terms of symptom expression and viral DNA movement. On the contrary, the CP7 mutant was unable to systemically infect tobacco plants, whereas only 10% of the plants inoculated with the CP191 mutant became infected. The CP7 mutant was complemented by coinoculating it with another geminivirus (taino tomato mottle virus). No complementation was observed in plants from nine transgenic tobacco lines expressing CP under the control of the cauliflower mosaic virus (CaMV) 35S promoter. However, 3 out of 10 lines expressing CP under the control of its own promoter (693 nucleotides) were able to complement the CP7 mutant. Interestingly, upon infection, the levels of CP mRNA in 693CP plants increased dramatically, probably due to transactivation of the CP promoter by the viral protein AC2.     

Resistance to Rhizoctonia solani AG‐2‐2 (IIIB) in creeping bentgrass plants transformed with pepper esterase gene PepEST

A pepper esterase (PepEST) gene was introduced into creeping bentgrass (Agrostis stolonifera) by Agrobacterium‐mediated transformation. Purified recombinant PepEST proteins were sufficient to inhibit the growth of the fungal pathogens Rhizoctonia solani AG2‐2 (IIIB) (causing brown patch) and Sclerotinia homoeocarpa (dollar spot), but not the oomycete responsible for pythium blight, Pythium aphanidermatum. PepEST proteins were most effective against R. solani. After genetic transformation of creeping bentgrass with PepEST, the genomic integration of transgenes bar and PepEST was confirmed by Southern blot analysis, and their expression was also validated by northern blot and western blot analyses. Disease severity on R. solani‐inoculated leaves of transgenic plants was <10% compared to ca. 50% in non‐transgenic plants. Microscopic observation of infected leaves indicated that PepEST inhibited the growth of hyphae upon fungal infection.