Virus-derived transgenes expressing hairpin RNA give immunity to Tobacco mosaic virusand Cucumber mosaic virus

Hu Qiong, Niu Yanbing, Zhang Kai, Yong Liu, Zhou, Zhou Xueping

Description

An effective method for obtaining resistant transgenic plants is to induce RNA silencing by expressing virus-derived dsRNA in plants and this method has been successfully implemented for the generation of different plant lines resistant to many plant viruses. Results Inverted repeats of the partial Tobacco mosaic virus (TMV) movement protein (MP) gene and the partial Cucumber mosaic virus (CMV) replication protein (Rep) gene were introduced into the plant expression vector and the recombinant plasmids were transformed into Agrobacterium tumefaciens . Agrobacterium -mediated transformation was carried out and three transgenic tobacco lines (MP16-17-3, MP16-17-29 and MP16-17-58) immune to TMV infection and three transgenic tobacco lines (Rep15-1-1, Rep15-1-7 and Rep15-1-32) immune to CMV infection were obtained. Virus inoculation assays showed that the resistance of these transgenic plants could inherit and keep stable in T 4 progeny. The low temperature (15℃) did not influence the resistance of transgenic plants. There was no significant correlation between the resistance and the copy number of the transgene. CMV infection could not break the resistance to TMV in the transgenic tobacco plants expressing TMV hairpin MP RNA. Conclusions We have demonstrated that transgenic tobacco plants expressed partial TMV movement gene and partial CMV replicase gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to TMV or CMV infection.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Hu et al. Virology Journal 2011, 8:41
http://www.virologyj.com/content/8/1/41
RESEARCH Open Access
Virus-derived transgenes expressing hairpin RNA
give immunity to Tobacco mosaic virus and
Cucumber mosaic virus
1,2 1 1 1 1*Qiong Hu , Yanbing Niu , Kai Zhang , Yong Liu , Xueping Zhou
Abstract
Background: An effective method for obtaining resistant transgenic plants is to induce RNA silencing by
expressing virus-derived dsRNA in plants and this method has been successfully implemented for the generation of
different plant lines resistant to many plant viruses.
Results: Inverted repeats of the partial Tobacco mosaic virus (TMV) movement protein (MP) gene and the partial
Cucumber mosaic virus (CMV) replication protein (Rep) gene were introduced into the plant expression vector and
the recombinant plasmids were transformed into Agrobacterium tumefaciens. Agrobacterium-mediated
transformation was carried out and three transgenic tobacco lines (MP16-17-3, MP16-17-29 and MP16-17-58)
immune to TMV infection and three transgenic tobacco lines (Rep15-1-1, Rep15-1-7 and Rep15-1-32) immune to
CMV infection were obtained. Virus inoculation assays showed that the resistance of these transgenic plants could
inherit and keep stable in T progeny. The low temperature (15℃) did not influence the resistance of transgenic4
plants. There was no significant correlation between the resistance and the copy number of the transgene. CMV
infection could not break the resistance to TMV in the transgenic tobacco plants expressing TMV hairpin MP RNA.
Conclusions: We have demonstrated that transgenic tobacco plants expressed partial TMV movement gene and
partial CMV replicase gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to
TMV or CMV infection.
Background mediated resistance in transgenic plants [1]. Pathogen-
The plant disease caused by Tobacco mosaic virus (TMV) derived resistance for CMV often showed only partial
or Cucumber mosaic virus (CMV) is found worldwide. resistance or very narrow spectrum of resistance to the
The two viruses are known to infect more than 150 spe- virus [2].
cies of herbaceous, dicotyledonous plants including many RNA silencing or post-transcriptional gene silencing
vegetables, flowers, and weeds. TMV and CMV cause (PTGS), developed during plant evolution, functions as
serious losses on several crops including tobacco, tomato, a defense mechanism against foreign nucleic acid inva-
cucumber, pepper and many ornamentals. During the sions (viruses, transponsons, transgenes) [3]. Since the
last decade, several laboratories have tried to introduce phenomenon of RNA silencing was first observed by
resistance to TMV or CMV by genetic engineering. Virus Napoli [4], research has been carried out to elucidate its
mechanism. PTGS is a mechanism closely related toresistance in plants containing virus-derived transgene,
usually by the expression of functional or dysfunctional RNA interference, which is involved in plant defense
coat protein, movement protein or polymerase gene, has against virus infection [5,6]. It was found that when a
been widely reported. The TMV coat protein gene was inverted repeated sequences of partial cDNA from a
used in the first demonstration of virus-derived, protein- plant virus are introduced into host plants for expres-
sion of dsRNA and induction of RNA silencing, the
transgenic plants can silence virus corresponding gene* Correspondence: [e-mail]
1State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang and are resistant to virus infection [7,8]. More than 90%
University, Hangzhou, 310029, P.R. China of transgenic Nicotiana benthamiana lines were
Full list of author information is available at the end of the article
© 2011 Hu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.Hu et al. Virology Journal 2011, 8:41 Page 2 of 11
http://www.virologyj.com/content/8/1/41
resistant to the virus when engineered with hairpin con- The T progenies of T parental lines, MP16, MP31,1 0
structs using Plum pox virus P1 and Hc-Pro genes MP39, MP53, Rep15, Rep17, Rep25 and Rep53 contained
sequences under the 35S-cauliflower mosaic virus pro- some plants that were immune and others that were sus-
moter [9]. For the current study, we expressed the partial ceptible, whereas the T parental line MP36 or Rep7270
TMV movement protein (MP) gene and the partial CMV which was susceptible to the virus yielded only suscepti-
replication protein (Rep) gene in the form of an intermo- ble progenies in successive generations (Table 1). The
lecular intron-hairpin RNA in transgenic tobacco. We progeny of T lines MP16 and Rep15 was confirmed to a0
analyzed the resistance of T to T transgenic plants. We have a segregation ratio of 3:1 (immune: susceptible),0 4
found that the two T transgenic lines with single copy suggesting the presence of a single dominant transgene4
were completely resistant to the corresponding virus, and locus in each line, and Southern blot analysis revealed
the viral resistance of transgenic plants did not be that the loci each appear to have a single transgene
affected by the low temperature (15℃). (Table 1).
Responses to TMV or CMV infection were further
Results examined for the phenotype of T,T and T genera-2 3 4
Transformation and analysis of T plants tion. Resistant T lines were randomly selected from0 1
Transgenic tobacco plants expressing hairpin RNA each of the six T lines (MP16, MP31, MP39, MP53,0
derived from TMV ΔMP or CMV ΔRep gene were gen- Rep15 and Rep17) that generated both resistant and sus-
erated by Agrobacterium tumefaciens-mediated transfor- ceptible progenies and the two T lines (MP36 or0
mation (Figure 1). Thirty T transgenic plant lines Rep727) that only generated susceptible progenies were0
containing TMV MP sequences and twenty T trans- also selected. In the screening of the T generation,0 2
genic plant lines containing CMV Rep sequences were plants were randomly selected and inoculated with
obtained by kanamycin selection. The specific DNA TMV or CMV. Most of the T generation plants derived2
fragment was amplified in all transgenic lines by PCR from resistant T lines segregated for both resistant and1
using primers TMV MP-F1 and TMV MP-R1 specific susceptible phenotype, whereas all T progenies from2
for TMV MP or primers ΔRep-F and ΔRep-R specific the resistant T lines, MP16-17 and Rep15-1, were1
for CMV Rep gene (data not shown). Southern blot ana- immune, which showed no any symptoms and no virus
lyses of selected transgenic lines indicated that the MP replication when measured by TAS-ELISA at 25 days
or Rep gene fragment was integrated into the genomic after inoculation (Table 2). The resistant T lines MP16-2
DNA and the copy number of the foreign gene was esti- 17-3, MP16-17-29, MP16-17-58, Rep15-1-1, Rep15-1-7
mated to be one to more than five (Table 1). and Rep15-1-32 generated only immune phenotypes in
thesuccessiveT and T generations, confirming the3 4
Resistant response of T to T transgenic progenies to stable inheritance of resistance (Table 2), although most0 4
infection of TMV or CMV of the other resistant parental T or T segregated for a2 3
The successive generation seeds were obtained by self- few susceptible plants in T or T generations. On the3 4
pollination from inoculated plants and the progenies of contrary, all of the T progenies from susceptible T2 1
different lines were gained simultaneously for further lines (MP36-17 or Rep727-1), were susceptible to TMV
analyses. Seedlings per each line were randomly taken or CMV and did not segregate for resistance in the suc-
from the resultant regenerates for virus inoculation tests. cessive generations (Table 2). T transgenic plants kept4
immunity phenotypes were shown in Figure 2. The
immunity transgenic plants (hp) were completely
asymptomatic (Figure 2A and 2B). When samples from
inoculated leaves and new emergent leaves of different
immune T lines were detected with TAS-ELISA at4
25 days after CMV or TMV inoculation, the absorbance
value from either inoculated or new (systemic) leaves of
inoculated plants were as low as negative samples (wt-)
(Figure 2C and 2D), which indicated that the virus repli-
cation was prevented at local and systemic infection in
transgenic immunity plants. Severe mosaic symptoms
were found at 30 days after TMV or CMV inoculation
Figure 1 (A) Schematic map of the T-DNA region of pBIN- on untransformed wild-type plants (wt+) (Figure 2A and
CMVΔRep(i/r) and (B) Diagram of self-complementary (hairpin) 2B). The results suggest that the resistance induced
RNA produced by pBIN-CMVΔRep(i/r). CaMV 35S: Cauliflower by the hairpin RNA is stably inherited through self-
mosaic virus 35S promoter; nos ter: nopaline synthase terminator.
pollination for the fourth generations.Hu et al. Virology Journal 2011, 8:41 Page 3 of 11
http://www.virologyj.com/content/8/1/41
Table 1 Testing of T and T transgenic plants for TMV or CMV resistance0 1
aT line number T reaction to TMV T reaction to CMV T copy number of transgene (Southern) T segregation immune:susceptible0 0 0 0 1
MP16 Immune 1 36:14*
MP31 >3 31:19
MP39 Immune