RNA interference based resistance against chilli leaf curl disease complex

Abstract

Chilli (Capsicum annuum), a member of the family Solanaceae, is an important spice crop cultivated in tropical and subtropical countries. Chilli leaf curl disease (ChLCD) is a limiting factor for chilli yield across Pakistan and India. Symptoms of ChLCD include severe upward leaf curl with cup-shape, yellowing and stunted plant growth. This disease is caused by begomoviruses (single-stranded DNA viruses (family Geminiviridae) that are transmitted by whiteflies). All three different types of begomoviruses are already reported from chillies. In this study chilli samples showing typical disease symptoms were collected from Faisalabad in the Province of Punjab (Pakistan) during the year 2006. All samples were positive for begomoviruses and Pepper leaf curl Lahore virus (PepLCLV) along with Tomato leaf curl New Delhi virus DNA B and Chilli leaf curl betasatellite (ChLCB) were identified. The DNA of Pepper leaf curl Lahore virus consisted of 2747 nucleotides and had the highest sequence identity (99%) with PepLCLV-[PK: Lah: 04] AM404179). Agrobacterium-mediated inoculation of the partial repeat construct of PepLCLV clone obtained in this study to Nicotiana benthamiana induced very mild symptoms and very low flow of viral DNA were detected in infected plant leaves. Co-inoculation of ChLCB with PepLCLV to N. benthamiana did not affect the symptoms severity or the virus titre. However neither the PepLCLV alone or with ChLCB was able to induce any symptoms on N. tabacum L. and C. annuum. Inoculation of PepLCLV with DNA B of ToLCNDV induced very severe symptoms in N. benthamiana, N. tabacum and symptoms typical of ChLCD in C. annuum. Southern hybridization analysis showed very high DNA accumulation for PepLCLV and DNA B of ToLCNDV in all three plant species. Sequence analysis showed that predicted rep-binding iterons in PepLCLV (GGGGAC) was different with two nucleotides from that of ToLCNDV DNA B (GGTGTC). This indicated tolerance of two nucleotide differences in iterated elements for replication. Based on this study, it is proposed that PepLCLV has been recently mobilized into chillies upon its interaction with DNA B of ToLCNDV. This is the first experimental demonstration of infectivity for a bipartite begomovirus causing ChLCD in chillies from Pakistan and suggests that component capture may contribute to the emerging complexity of begomovirus diseases in the region. NIBGE |School of Biotechnology NIBGE Faisalabad [QAU Islamabad] iv The purpose of this study was to develop a broad-spectrum resistance against ChLCD complex based on the concept of pathogen-derived resistance. A hairpin RNAi construct (peAC1-AC2dsRNA/pFGC) based on overlapping region of highly conserved region of Rep and TrAP of PepLCLV was produced in a binary vector pFGC5941. In order to study silencing efficiency of peAC1-AC2dsRNA/pFGC, the construct was transiently challenged with PepLCLV along with DNA B ToLCNDV. Results showed that the RNAi construct was successful in blocking the viral infection as all tested plants were symptomless. Transgenic tobacco plants expressing this construct challenged with PepLCLV and DNA B of ToLCNDV by agroinoculation and with viruliferous whiteflies showed variable resistance ranging from 6.6% to 93.3%. Lines showing resistance more than 75% were ranked resistant/tolerant while lines showing resistance less than 50% were ranked susceptible. One line TA14 showing 93.3% was ranked as highly resistant/tolerant while the line TA 3.2 showing 6.6 % resistance/tolerance was ranked as highly suscepteible. These lines also exhibited significant resistance against ToLCNDV. The relatively conserved nature of Rep and TrAP and their ability to help in development of resistance against heterologous virus suggested that the technology may be useful to develop broad-spectrum resistance. Plants need broad spectrum resistance because they were often infected with multiple begomoviruses in the field. Some viral proteins interfere with different cell signalling pathways and induce symptoms in plants. For example expression of P6 protein of CaMV in Arabidopsis induced dwarfness in transgenic plants. It is reported that Arabidopsis plants with TIR3 gene mutated (tir3) are also dwarf. P6 transgenic (A7, B6) and tir3 Arabidopsis plants which were resistant to auxin and ethylene also showed resitance to 2,3,5-Triiodobenzoic Acid (TIBA) treatment. It indicates that P6 interacts with a pathway overlapped with TIR pathway. Symptoms in Arabidopsis expressing the P6 protein of CaMV probably comes by disturbance of auxin response factor 10 (ARF10), ARF16, and ARF17 also. Also P6-expressing transgenic Arabidopsis plants showed reduced accumulation of miR160 which is known to regulate ARF10, ARF16 and ARF17. A protocol was also developed for chilli plant regeneration using hypocotyl and cotyledon explants. The study was conducted to observe the effect of genotypes, culture conditions and growth regulators on plant regeneration of chili pepper (C. NIBGE |School of Biotechnology NIBGE Faisalabad [QAU Islamabad] v annuum) genotypes grown in Pakistan including Seedex Pepper (SP), Loungi, Tatapuri and Sanam. Of the evaluated genotypes, SP was found to be the most responsive for both hypocotyl and cotyledon explants. Hypocotyl and cotyledon explants were tested for transformation by A. tumefactions LBA4404 having the 35S GFP/pFGC construct and A. tumefaciens EHA105 with peAC1-AC2dsRNA/pFGC construct. Co-cultivation at different temperatures (22 and 25ºC), photoperiods (16h light 8h dark, and complete darkness) as well as co-cultivation time periods, were evaluated. GFP assays showed that putative transgenic calli had not been transformed and calli died after 40-60 days. The experiment was repeated ten times. The data presented in this thesis should help in devising novel control strategies against Begomoviruses. A combination of novel sources of resistance with natural sources of resistance may help to exploit the technology in the field conditions. However, because most pepper varieties are recalcitrant to genetic transformation, control of diseases caused by the ChLCD complex using this strategy awaits future progress.