Artificial micro RNA (amiRNA)-mediated resistance against whitefly (Bemisia tabaci) targeting three genes

Abstract

Whitefly (Bemicia tabaci) is an insect pest that causes severe losses in economically important crops by sucking the plant sap and transmitting plant viruses. We used the promising new approach of plant-mediated artificial miRNA (amiRNA) expression against three essential genes of whitefly. The Arabidopsis thaliana miR159 precursor was modified and engineered to express artificial miRNAs (amiRNAs) targeting three vital genes of whitefly, sex lethal (Sxl) protein, acetylcholinesterase (AChE) and orcokinin (Orc). The amiRNA^WF construct was transformed into the Nicotiana tabacum plants. Whiteflies were infested in separate cages on both control and transgenic plants. After four weeks of infestation, the nymphs were counted and real time quantitative PCR (RT-qPCR) was performed to assess the relative expression of whitefly genes. The transgenic N. tabacum plants showed resistance against whitefly and the number of whiteflies in the next generation were substantially reduced on transgenic plants compared to control plants. Abnormal egg hatching and poor development was observed and only a small number of whitefly nymphs matured to adults. RT-qPCR results indicated that the expression of target genes (Sxl, AChE and Orc) in the nymphs was considerably down-regulated in whiteflies reared on transgenic plants. The amiRNA mediated resistance against whitefly in transgenic plants may pave the way for engineering resistance against whitefly in cotton plants.

Introduction

Whitefly (Bemisia tabaci) belongs to the family Aleyrodidae, order Hemiptera [suborder Homoptera] (Mound, 1984). Whitefly is one of the major insect pest of vegetables, cotton and many other economically vital crops worldwide. Whitefly is also the vector for several economically important plant viruses including begomoviruses. Begomovirus belongs to family Geminiviridae. Whitefly feed on wide host range of plants, feeding on more than 500 plant species of 74 families (Martin, 1999; Perring, 2001). Whiteflies like aphids and other insects belong to the order Hemiptera and feed on plant sap. Whiteflies have a very unique haplodiploid genome where males have haploid genome produced from unfertilized eggs and inherit only the maternal genome, while female offspring has diploid genome produced from fertilized eggs and they carry genomes from both parents (Blackman and Cahill, 1998; Schrader, 1920). There are three main types of whitefly known as A biotype,B biotype and Q biotype (Brown et al., 2000; Abdullah et al., 2006; Bayhan et al., 2006; Brown, 2007). The whitefly complex is composed of 34 different species varying biologically and genetically (De Barro et al., 2011; Boykin et al., 2012a, 2012b) which form 12 major phylogenetic groups (De Barro et al., 2011; Boykin and De Barro, 2014; Boykin et al., 2007).

The whitefly cryptic species complex causes severe damage to the fiber and food crops worldwide (Pimentel et al., 2005) and is listed in the top 100 of the world's worst invasive alien species (http://www.iucngisd.org/gisd/100_worst.php). . Whitefly feed abaxially on the leaf and damages the host plants by sucking plant sap and depriving the plant of essential nutrients (Martin, 1999; Perring, 2001). Whiteflies also produce honeydew in large quantity which favors the growth of fungus on leaves and reduce the photosynthetic activity of plants (Brown et al., 1995). Whiteflies also damage the plants by transmitting plant viruses in many crops including cotton, cassava, tobacco, sweet potatoes, chillies, beans and tomatoes. Viral diseases of plants caused by geminiviruses that are vectored by whiteflies are of great concern and result in great losses in crops (Brown et al., 1995).

RNA interference (RNAi) is induced by double stranded RNA (dsRNA), resulting in silencing of the target gene in highly sequence-specific manner. RNAi is a conserved mechanism known as quelling in fungi, post-transcriptional gene silencing (PTGS) in plants, and RNAi in animals (Bisaro, 2006; Baulcombe, 2005; Fire et al., 1998). Most living organisms possess RNAi machinery, and when they are challenged by foreign pathogens, RNAi may play a role in the defense response (Meister and Tuschl, 2004; Mello and Conte, 2004). RNAi based resistance strategies have been demonstrated against sucking pests such as whitefly and aphid. Various RNAi strategies such as sense or antisense RNA, hairpin RNA or dsRNA, and artificial microRNA (amiRNA) have been exploited to induce RNAi-mediated gene silencing in sucking pests. Hairpin dsRNA is delivered to the insects through an oral route via artificial diet or insects fed on transgenic plants expressing dsRNA. A recent study showed that oral delivery of synthetic dsRNA resulted in significant knock down of genes expressing exclusively in the whitefly gut (Vyas et al., 2017). A plant-mediated dsRNA expression approach was used for other studies to knock down the expression of a number of important genes of whitefly and aphid (Raza et al., 2016; Malik et al., 2016; Thakur et al., 2014). The plant-mediated artificial microRNA expression approach has been used to silence MpAChE2 gene of aphid (Myzus persicae) (Guo et al., 2014).

Here, we stably expressed artificial micro RNAs (amiRNAs) in planta against three important whitefly genes to create resistance against whitefly. As previous studies show that by changing several nucleotides within a miRNA backbone does not disturb its biogenesis (Schwab et al., 2006; Ossowski et al., 2008). This possibility to alter 21-nt miRNA sequence was harnessed to target desired genes of whitefly. The backbone of pre-miRNA159^a of A. thaliana was used to generate pre-amiRNAs^WF containing sequences against three vital genes of whitefly. Compared to the RNAi effect of long hairpin dsRNA and VIGS systems, plant generated amiRNAs have several advantages. The plant generated amiRNAs have minimal predicted off-target effects and have the ability to target multiple genes (Ossowski et al., 2008; Molnar et al., 2009). The silencing activity of amiRNAs is stable through to several generations (Molnar et al., 2009). It has also been suggested that the amiRNAs have fewer environmental and biosafety problems in agriculture compared to other resistant strategies (Duan et al., 2008; Liu and Chen, 2010). The amiRNAs approach also have the advantage to allow the creation of constructs of multiple amiRNAs targeting multiple genes or alleles (Lin et al., 2009; Niu et al., 2006).

In this study three genes of whitefly named as Sex-lethal (Sxl), Acetylcholinesterase (AChE) and Orcokinin (Orc) were selected to develop an amiRNA^WF construct for resistance against whiteflies. Whiteflies exhibit a haplo-diploid sex determination system (Blackman and Cahill, 1998) and the sex determination is not well understood in hemipterans like aphids and whiteflies. The model insects such as Drosophila melanogaster and Apis mellifera are used as model for understanding the molecular basis of sex determination system in whiteflies. Sxl is an important gene of the sex determination cascade in D. melanogaster. AChE gene functions as a neurotransmitter and catalyzes the hydrolysis of acetylcholine into its acetyl-CoA and acetate components. The blockage of AChE results in increased levels of acetylcholine which causes muscular dysfunction, paralysis and ultimately death of insects (Olivera et al., 2003; Shapira et al., 2001). Orc gene code for myotropic neuropeptides which regulate various physiological and developmental processes (Nässel and Winther, 2010). The Orc appears to regulate the function of circadian clock that controls locomotory activity in insects (Hofer and Homberg, 2006).

The candidate genes Sxl, AChE and Orc were selected from all available nucleotides, unigenes, and ESTs of drosophila, aphid and A. mellifera. Efficacy of the construct was tested on both transgenic and control plants by infesting them with whiteflies for four weeks. The relative gene expression was measured with real time quantitative polymerase chain reaction (RT-qPCR) amplification in the next generation of whitefly nymphs. The gene expression profiling results using RT-qPCR showed that the expression of all three target genes was substantially down-regulated in whiteflies feeding on transgenic plants. There was abnormal nymph development and the whitefly population was substantially reduced on transgenic plants compared to the control plants. Plant expression of whitefly specific amiRNAs provide a promising new tool to develop whitefly resistance in natural hosts such as cotton plants.