Analysis of transitive RNA silencing after grafting in transgenic plants with the coat protein gene of <Emphasis Type="Italic">Sweet potato feathery mottle virus</Emphasis>

We have previously reported the graft transmission of target specificity for RNA silencing using transgenic Nicotiana benthamiana plants expressing the coat protein gene (CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Transgenic plants carrying the 5′ 200 and 400 bp regions of CP were newly produced. From these plants, two silenced and two non-silenced lines were selected to investigate the manifestation of transitive RNA silencing by graft experiments. Non-silenced scions carrying the entire transgene were grafted onto either 5′ or 3′ silencing inducer rootstocks. When non-silenced scions were grafted onto 5′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions and spread toward the 3′ region of the transgene mRNA. Similarly, when non-silenced scions were grafted onto 3′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions, but was restricted to the 3′ region of the transgene and did not spread to the 5′ region. In addition, results from crossing experiments, involving non-silenced and 3′ silencing inducer plants, confirmed the above finding. This indicates that RNA silencing spreads in the 5′–3′ direction, not in the 3′–5′ direction, along the transgene mRNA.     

Grafting on a Non-Transgenic Tolerant Tomato Variety Confers Resistance to the Infection of a Sw5-Breaking Strain of Tomato spotted wilt virus via RNA Silencing

RNA silencing controls endogenous gene expression and drives defensive reactions against invasive nucleic acids like viruses. In plants, it has been demonstrated that RNA silencing can be transmitted through grafting between scions and silenced rootstocks to attenuate virus and viroid accumulation in the scions. This has been obtained mostly using transgenic plants, which may be a drawback in current agriculture. In the present study, we examined the dynamics of infection of a resistance-breaking strain of Tomato spotted wilt virus (RB-TSWV) through the graft between an old Apulian (southern Italy) tomato variety, denoted Sl-Ma, used as a rootstock and commercial tomato varieties used as scions. In tests with non-grafted plants, Sl-Ma showed resistance to the RB-TSWV infection as viral RNA accumulated at low levels and plants recovered from disease symptoms by 21 days post inoculation. The resistance trait was transmitted to the otherwise highly susceptible tomato genotypes grafted onto Sl-Ma. The results from the analysis of small RNAs hallmark genes involved in RNA silencing and virus-induced gene silencing suggest that RNA silencing is involved in the resistance showed by Sl-Ma against RB-TSWV and in scions grafted on this rootstock. The results from self-grafted susceptible tomato varieties suggest also that RNA silencing is enhanced by the graft itself. We can foresee interesting practical implications of the approach described in this paper.     

Rootstock‐to‐scion transfer of transgene‐derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry

Small interfering RNAs (siRNAs) are silencing signals in plants. Virus‐resistant transgenic rootstocks developed through siRNA‐mediated gene silencing may enhance virus resistance of nontransgenic scions via siRNAs transported from the transgenic rootstocks. However, convincing evidence of rootstock‐to‐scion movement of siRNAs of exogenous genes in woody plants is still lacking. To determine whether exogenous siRNAs can be transferred, nontransgenic sweet cherry (scions) was grafted on transgenic cherry rootstocks (TRs), which was transformed with an RNA interference (RNAi) vector expressing short hairpin RNAs of the genomic RNA3 of Prunus necrotic ringspot virus (PNRSV‐hpRNA). Small RNA sequencing was conducted using bud tissues of TRs and those of grafted (rootstock/scion) trees, locating at about 1.2 m above the graft unions. Comparison of the siRNA profiles revealed that the PNRSV‐hpRNA was efficient in producing siRNAs and eliminating PNRSV in the TRs. Furthermore, our study confirmed, for the first time, the long‐distance (1.2 m) transfer of PNRSV‐hpRNA‐derived siRNAs from the transgenic rootstock to the nontransgenic scion in woody plants. Inoculation of nontransgenic scions with PNRSV revealed that the transferred siRNAs enhanced PNRSV resistance of the scions grafted on the TRs. Collectively, these findings provide the foundation for ‘using transgenic rootstocks to produce products of nontransgenic scions in fruit trees'.     

转录后基因沉默--植物抵御外来病毒入侵的一种机制

基因沉默是近几年来在转基因植物中发现的一种后生遗传现象。基因沉默大体可以分为两类：位置效应引起的基因沉默和同源依赖的基因沉默。其中,同源依赖的基因沉默又可以分为转录水平的基因沉默和转录后水平的基因沉默。基因沉默的发现使得人们对植物和病毒的相互关系有了一个新的认识。基因沉默研究中所发现的转录后基因沉默现象是植物抵御病毒入侵、保持自身基因组完整性的一种防御机制,是植物与病毒共进化的结果。对于沉默产生的机理,尤其是转录后基因沉默,已经提出不少模型,但是都未能较全面地解释基因沉默中出现的各种实验现象。该文现就实验所取得的相关结果、转录后基因沉默机制和植物对病毒防御机制的相互关系,以及其研究进展进行综述。     

Conferring high‐temperature tolerance to nontransgenic tomato scions using graft transmission of RNA silencing of the fatty acid desaturase gene

We investigated graft transmission of high‐temperature tolerance in tomato scions to nontransgenic scions from transgenic rootstocks, where the fatty acid desaturase gene (LeFAD7) was RNA‐silenced. Tomato was transformed with a plasmid carrying an inverted repeat of LeFAD7 by Agrobacterium. Several transgenic lines showed the lower amounts of LeFAD7 RNA and unsaturated fatty acids, while nontransgenic control did not, and siRNA was detected in the transgenic lines, but not in control. These lines grew under conditions of high temperature, while nontransgenic control did not. Further, the nontransgenic plants were grafted onto the silenced transgenic plants. The scions showed less of the target gene RNA, and siRNA was detected. Under high‐temperature conditions, these grafted plants grew, while control grafted plants did not. Thus, it was shown that high‐temperature tolerance was conferred in the nontransgenic scions after grafting onto the silenced rootstocks.     

Expression of Pokeweed Antiviral Protein in Transgenic Plants Induces Virus Resistance in Grafted Wild-Type Plants Independently of Salicylic Acid Accumulation and Pathogenesis-Related Protein Synthesis

Pokeweed antiviral protein (PAP), a 29-kD protein isolated from Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. Transgenic tobacco (Nicotiana tabacum) plants expressing PAP or a variant (PAP-v) were shown to be resistant to a broad spectrum of plant viruses. Expression of PAP-v in transgenic plants induces synthesis of pathogenesis-related proteins and a very weak (<2-fold) increase in salicylic acid levels. Using reciprocal grafting experiments, we demonstrate here that transgenic tobacco rootstocks expressing PAP-v induce resistance to tobacco mosaic virus infection in both N. tabacum NN and nn scions. Increased resistance to potato virus X was also observed in N. tabacum nn scions grafted on transgenic rootstocks. PAP expression was not detected in the wild-type scions or rootstocks that showed virus resistance, nor was there any increase in salicylic acid levels or pathogenesis-related protein synthesis. Grafting experiments with transgenic plants expressing an inactive PAP mutant demonstrated that an intact active site of PAP is necessary for induction of virus resistance in wild-type scions. These results indicate that enzymatic activity of PAP is responsible for generating a signal that renders wild-type scions resistant to virus infection in the absence of increased salicylic acid levels and pathogenesis-related protein synthesis.     

Detection of Grapevine Viruses in Poland

During a 3‐year study, grapevines from 23 vineyards in Poland were surveyed for virus diseases and tested to determine the prevalence of the most economically important viruses by RT‐PCR. The rate of positive samples was 2.2% for grapevine leafroll‐associated virus 1 (GLRaV‐1), 1.9% for grapevine leafroll‐associated virus 2 (GLRaV‐2), 1.5% grapevine leafroll‐associated virus 3 (GLRaV‐3), 1.9% for grapevine virus A (GVA), 0.2% for grapevine virus B (GVB), 0.2% for grapevine virus E (GVE), 0.65% for grapevine fanleaf virus (GFLV), 20.4% for grapevine fleck virus (GFkV) and 71.9% for grapevine rupestris stem pitting‐associated virus (GRSPaV). These viruses were found to occur as single or mixed infections of different combinations in individual grapevines. The overall viral infection rate in the surveyed grapevines was 82.6%. GRSPaV is the most widely distributed virus of all the viruses currently detected in the region. DNA sequencing confirmed the identification of the viruses in selected samples, and analysis indicated that the Polish isolates shared a close molecular identity with the corresponding isolates in GenBank. To our knowledge, this is the first detection of GLRaV‐1, ‐2, ‐3, GVA, GVB, GVE, GFLV, GFkV and GRSPaV in Poland.     

Agrobacterium rhizogenes—transformed plant roots as a source of grapevine viruses for purification

Agrobacterium rhizogenes-mediated transformation was applied toVitis spp. andNicotiana spp. infected by different grapevine phloem-limited viruses (grapevine fleck virus, grapevine virus A, grapevine virus B) to obtain root cultures for virus purification. All plant species were successfully transformed, and several clones were established in liquid culture. Transformed grapevine roots contained as much virus as non transformed roots and more than leaves, as assessed by ELISA and thin sectioning. Likewise, transformed roots ofNicotiana benthamiana Domin. contained in average more GVA than leaves, especially those at the base and the top of the plant, whereas withNicotiana occidentalis wheel., GVB was apparently less concentrated than in leaves.Nicotiana root grew faster than those ofVitis. All viruses multiplied and persisted in root cultures, which were successfully used for purification. Virus yields were the same (GFkV and GVB) or higher (GVB) than those reported in the literature. Grapevine roots may prove useful for culturing and purifying other non-mechanically transmissible grapevine viruses.