Transgenes in plants: protection against viruses and insects

Protective transgenes introduced into plants can be classified as directed against insects, fungi, bacteria or viruses. Mechanisms by which they protect plants in some cases are relatively simple and understood while in most cases they present only the field of rapidly progressing investigations. A brief review of the recent concepts of the resistance induced in plants against viruses by virus-derived transgenes is presented with emphasising the RNA mediated resistance. The RNA mediated resistance seems to operate in Nicotiana benthamiana plants transformed in our laboratory with cDNA of the PPV CP gene: both translatable and untranslatable versions of the cDNA made the transformed plants resistant against PPV. The resistant plants contained more than one copy of the transgene. To protect against insects plants were in our laboratory transformed with potato proteinase inhibitor II gene (PPI-II). The PPI-II gene expressed in model plants inhibited trypsin activity to an expected level.     

CMV抑制PVYN CP基因沉默及其介导的抗病性

以前曾报道用RNA介导的抗病毒策略,获得了高度抗病的表达马铃薯Y病毒坏死株系外壳蛋白基因(PVY^N CP)的转基因烟草,并对T1、T2代转基因植株进行了遗传和抗病性分析。此次以T,代转基因植株为试验材料,在筛选高度抗病植株并证明其抗病性是基于转基因沉默的基础上,采用Northern杂交的方法,证明CMV侵染抑制了转基因植株中PVY^N CP基因的沉默,而且CMV对PVY^N CP基因沉默的抑制部位是发生在接种后的新生叶上,接种叶及其下部叶片中PVY^N CP基因沉默则未受到影响。采用ELISA方法对CMV PVY^N复合接种的转基因植株进行PVY^N检测,结果表明,接种叶及下部叶没有检测到PVY^N,植株叶片对PVY^N表现为抗病。而在CMV接种后植株新生叶中则检测出了高滴度的PVY^N,植株叶片对PVY^N表现为感病。该文报道了在表达PVY^N CP基因的RNA介导抗性转基因植株中,异源病毒侵染抑制了转基因的沉默,并导致转基因植株的抗病性丧失。     

Transgenic <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> plants resistant to cucumber green mottle mosaic virus based on RNA silencing

RNA silencing technology was used to confer resistance to cucumber green mottle mosaic virus (CGMMV). Nicotiana benthamiana was transformed with a transgene designed to produce an inverted repeat RNA containing CGMMV-coat protein gene (CP) sequences, which were separated by an intron sequence, under the control of the cauliflower mosaic virus 35S promoter. We attempted to confirm the resistance of seven independent transgenic lines; five lines showed resistance to CGMMV infection. The systemic spread of virus was prevented after the inoculation of CGMMV, and the CP-specific short interfering RNA (siRNA) was detected in resistant lines. Thus, the resistance against CGMMV through RNA silencing is strong and efficient.     

Rapid production and field testing of homozygous transgenic tobacco lines with virus resistance conferred by expression of satellite RNA and coat protein of cucumber mosaic virus

A procedure for the fast production of homozygotic transgenic plants was developed. Leaf discs of haploid tobacco plants from anther cultures were transformed with a chimaeric vector containing coat protein (CP) and satellite RNA (Sat-RNA) genes from cucumber mosaic virus (CMV). One-hundred-and-twelve Kanamycin-resistant transformed haploid plants were subjected to selection based on the expression of both CP and Sat-RNA. Eighty-nine transgenic plants expressing both genes were selected and tested for their resistance to CMV by inoculation with high concentration of CMV (200 g ml^–1). Only five plants showed no symptoms of viral infection 30 days after inoculation. These plants were then diploidized by colchicine treatment. Three homozygous diploid lines with high levels of resistance to CMV were obtained after only one generation. The three transgenic lines were further tested under field conditions. The results showed that the progenies of these transgenic lines were homozygous and were highly resistant to CMV under natural field infection and manual inoculation conditions.     

Resistance to Cucumber mosaic virus in Gladiolus plants transformed with either a defective replicase or coat protein subgroup II gene from Cucumber mosaic virus

Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These plants were multiplied in vitro and challenged with purified CMV isolated from Gladiolus using a hand-held gene gun. Three out of 19 independently transformed plants expressing the replicase gene under control of the duplicated CaMV 35S promoter were found to be resistant to CMV subgroup I. Three out of 21 independently transformed plants with the CMV subgroup II coat protein gene under control of the Arabidopsis UBQ3 promoter were resistant to CMV subgroup II. Eighteen independently transformed plants with either the CMV subgroup I coat protein or a combination of CMV subgroups I and II coat proteins were challenged and found to be susceptible to both CMV subgroups I or II. Virus resistant plants with the CMV replicase transgene expressed much lower RNA levels than resistant plants expressing the CMV subgroup II coat protein. This work will facilitate the evaluation of virus resistance in transgenic Gladiolus plants to yield improved floral quality and productivity.     

Development of resistant transgenic soybeans with inverted repeat-coat protein genes of soybean dwarf virus

In an attempt to generate soybean plants resistant to soybean dwarf virus (SbDV), we transformed a construct containing inverted repeat-SbDV coat protein (CP) genes spaced by β-glucuronidase (GUS) sequences into soybean somatic embryos via microprojectile bombardment. Three T₀ plants with an introduced CP gene were obtained, and one generated T₁ seeds. The presence of the transgene in T₁ plants was confirmed by PCR and Southern blot hybridization analysis, but expression of CP was not detected by northern blot hybridization analysis. Two months after inoculation of SbDV by aphid, T₂ plants contained little SbDV-specific RNA and remained symptomless. These plants contained SbDV-CP-specific siRNA. These results suggest that the T₂ plants achieved resistance to SbDV by an RNA-silencing-mediated process.     

Coat protein-mediated resistance to Turnip mosaic virus in oilseed rape (Brassica napus)

Oilseed rape (Brassica napus) lines transformedwith the coat protein (CP) gene of Turnip mosaic virus(TuMV) were used to determine the effectiveness of resistance to TuMV mediatedby CP RNA or coat protein. Lines with one, two, or more copies of transgeneswere produced. T₂ and T₃ lines containing the CP genewitha functional start codon synthesised coat protein and showed high, but variablelevels of resistance to TuMV (21–96% resistant plants per line). TheT₁ and T₂ progeny of all lines carrying the CP gene withamutated start codon so that RNA but not protein was expressed, were assusceptible to TuMV as controls. Thus, in these experiments we were able toinduce CP-mediated resistance, but not RNA-mediated resistance.     

RNA Silencing Mediated by Direct Repeats in Maize: A Potential Tool for Functional Genomics

It has been shown in tobacco and Arabidopsis that transgenes with multiple direct repeats induce RNA silencing at high frequency. In this study, we tried to establish a direct repeat-induced RNA silencing system in maize and evaluate whether it can be developed as a high throughput tool for functional genomics. Our results showed that the construct phC4, which carries four direct repeats of a chloramphenicol acetyl-transferase (CAT) gene, was able to induce silencing of itself with high efficiency in maize. Using a transient expression system, we further demonstrated that construct phC3G with a β-glucuronidase (GUS) gene located downstream of three direct repeats of CAT gene silenced not only itself in maize calli but also an “endogenous” GUS gene, which was stably expressed in maize calli. Most importantly, when constructs with the maize iojap (ij) gene inserted in either sense or antisense orientation into the downstream of four direct repeats of CAT gene were transformed into maize plants, co-suppression of endogenous and transgenic ij genes was detected in majority of transgenic maize plants. Our co-suppression results suggest that with improvements, this new approach has the potential to become an efficient research tool for high throughput functional genomics.