Resistance to Chilliveinal mottle virus (ChiVMV) in Nicotiano benthamiana Transformed with CP gene

Abstract

Chilli veinal mottle virus (ChiVMV, genus Potyvirus) is a common, wide spread, destructive and economically important plant virus that cause systemic infection in chilli resulting in mottling, vein clearing and leaf distruction. The successful production of chilli crop in Pakistan is mainly hampered due to ChiVMV which resulted in quite low average yield as compared to other countries. In Pakistan, limited work is only confined to occurrence and prevalence of ChiVMV from capsicum crop but no systemic and molecular study has been conducted on this virus. Development of control strategies and/or eliminate of viral diseases in plants requires better understanding and knowledge on the molecular characterization including sequencing and genetic diversity. Pathogen derived resistance (PDR) is one of the important strategies to implement effective and sustainable control measures against major viral diseases. The present research work was conducted to elucidate the genetic variability of Pakistani ChiVMV isolate and study the role of CP gene in conferring the resistance against Pakistani isolate of ChiVMV in model plant, Nicotiana benthamiana. In this regard Nicotiana benthamiana plants were transformed by using Agro-infiltration and stable transformation methods. Infectivity assay was done to evaluate the resistance level in transformed plants. Local lesion assay and host range studies were performed for biological purification, propogation and to evaluate the response of Pakistani ChiVMV isolate to different hosts. The 864 bases of CP gene was amplified in RT-PCR. CP cDNA was successfully cloned in pTZ57R/T TA cloning vector (p01). CP gene specific primers was designed with BglII and BsteII restriction sites. Using the clone p01 as template, 1kb fragment of CP gene was cloned with restriction sites (clone p02). CP cDNA from p02 was inserted in pcambia1301 after digestion with BglII and BsteII in sense or anti-sense direction under the control of CaMV35S promoter and transformed into Agrobacterium strain LBA4404. This transformed Agrobacterium culture was used for tansforming the Nicotiana benthamiana plants through agro-infiltration and by stable transformation methods. Confirmation of the transformation was carried out through PCR amplification of CP and hygromycin gene. Resistance level in transformed Nicotiana benthamiana plants against ChiVMV isolate ATIPK was confirmed through infectivity assay. Local lesions were produced by Pakistani ChiVMV isolate in Chenopodium quanoa plant. Different host plants like chilli varities, Datura metal and Nicotiana tabaccum showed the same symptoms for ChiVMV isolate ATIPK as reported for other ChiVMV isolates from the different areas of the world. A total of 986 nucleotides were obtained from each clone consisting of 96 basis of 3’ end of Nib gene, full length CP gene (864) and 23 bases from the 3’UTR. The sequence of ChiVMV(ATIPK) isolate was deposited to Genebank (acc. No. KJ472764). A pairwise comparison of ATIPK CP sequence with 22 highly matched sequences from database revealved that it has the highest nucleotide identity with an isolate from Thailand DQ854956 i.e 88.4% and lowest nucleotide identity was 86.8% with an isolate from India (DQ854964). Whereas highest amino acid identity was 91.8% with 3 isolates from India (EF213679, EF213681 and EF213703) and lowest amino acid identity was 89% with an isolate from Indonasia (AB703256). Putative transgenic N. benthamiana plants using CP gene of Pakistani ChiVMV isolate ATIPK, were used for the evaluation of resistance level against same isolate. Inoculated 2nd generation plants (T1-T8) indicated the presence of CP gene by overcome the infection and further confirmed through DAS ELISA test. The ELISA readings confirmed the phenotypic symptoms and indicated the presence of less amount of titer in transgenic Nicotiana benthamiana plants as compare to control plants. These findings on characterization and expression of understudied gene may enable us to define the position and role of it in the resistance that would be useful for developing effective control measures against this menaces threat (ChiVMV) to the solanacious crops.