Approaches for insect control Biological control Natural host resistance Control through chemical pesticides Genetically-engineered resistance

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1 Use of PVX vector as expression system and mealybug as a model target insect to resistance against sucking pests Muhammad Ashfaq The real challenge is sucking pests Mealybug Whiteflies Aphids Jassids Thrips Post-Bt Scenario M. Shaheen, A. M. Khan, S. Zaid, A. Raza, M. Ashfaq, I. Amin, Z. Mukhtar, S. Mansoor, Approaches for insect control ü Biological control ü Natural host resistance ü Control through chemical pesticides ü Genetically-engineered resistance Genetically engineered resistance Proteins and RNAi Bt VIPs Lectins Animal origin (Hvt etc) RNAi Application approaches Virus vector Feeding Transgenic plants In vitro synthesis of dsrna RNAi can be achieved by dsrna! Injection Ingestion Soaking Transgene Transgenic vectors 1

2 Insect control???? RNAi and insects Drosophila melanogaster (Kennerdell and Carthew, 1998) Anopheles gambiae (Paskewitz et al., 2006) Caterpillar Manduca sexta (Vermehren et al., 2001) Milkweed bug Oncopeltus fasciatus (Hughes and Kaufman, 2000) German Cockroach Blatella germanica (Cruz et al., 2006) Triatomine bug Rhodnius prolixus (Araujo et al., 2006) Apple brown moth Epiphyas postvittana (Turner et al., 2006) Ladybird beetle Harmonia axyridis (Kuwayama et al., 2006) Pea aphid Acyrthosiphon pisum (Mutti et al., 2006) Reported issues with insects RNAi varies among insect orders and species Insects lack RdRP Drosophila lacks sid-1 gene Sid-1 homologues identified in a number of insect species Another mechanism, receptor-mediated endocytosis By injection RNAi-mediated gene knockdown reported in: Diptera Coleoptera Hymenoptera Orthoptera Blattodea Lepidoptera Hemiptera Beye et al Specific developmental gene silencing in the honey bee using a homeobox motif Gene: E30 homeobox motif Injections of dsrna with an Oxford micromanipulator, a microinjector, and an ordinary stereomicroscope. Phenotypes of parental RNAi (parnai) and embryonic RNAi (emrnai) against Gryllus bimaculatus caudal (Gb cad). (a) Eggs obtained by parnai against Gb cad in which only the head region is formed (also see d and d ), indicating that the expression of Gb cad in the caudal region of the embryo is required for the formation of regions other than the head (Shinmyo et al. unpublished). (Mito et al. 2011). The phenotype of nymphal RNAi (nyrnai) against Gryllus bimaculatus laccase 2 lacks tanning (top). This result indicates that the expression of laccase 2 is crucial for cuticle tanning in G. bimaculatus. After injection of the dsrna for laccase 2 into nymphs at the fifth instar, the nymphs were reared to be adults. The bottom shows a control cricket. Bar represents 1 cm. (Mito et al. 2011) 2

3 3/8/11 By feeding By transgene Target genes: Cell-1 Hex-2 Zhou et al IMB Can we engineer insect resistance by expression of insect-specific double stranded RNA in plants??? Use of hairpin transgene in the host Putting RNAi in plants Use of dsrna for RNAi?? Use of hairpin transgenes in the host ü Control of coleopteran insects through RNAi Baum et al. Nature Biotechnology November, 07 The approach Developed Western corn borer cdna library Selected 290 target genes 125 showed significant mortality at 52ng/cm 67 showed mortality at 5.2 ng/cm 14 at less than 5.2 ng/cm 3

4 Plants expressing a V-ATPase A dsrna are protected from feeding damage Silencing bollworm P450 by plant mediated RNAi (Gossypol) Mao et al Objectives Ø To establish the use of a plant virus vector for expression of insecticidal genes by expression of cry1ac or HVt and Lectin from PVX. Ø To target insecticidal gene constructs based on RNA silencing from PVX vector and check insecticidal activity. Ø To determine the target specificity by checking some of the non target insects for insecticidal gene construct. Idea behind using PVX vector Potato Virus X is used as a screening tool for expression of insecticidal protein in phloem of the model plants to specifically target the sucking insects because PVX replicates and moves systemically through the phloem. PVX a single stranded RNA virus being used to trigger a plant mediated Systemic RNAi in insects via feeding sirna homologous to insect genes generated by the plants in response to viral induced gene silencing. Methods PVX (pgr107) transformed in Agrobacterium strain GV3101. Tobacco plants inoculated through infiltration and on symptoms arrival on plants to get VIGS well established phenomena. Bioassay on Mealybug conducted to reveal RNAi trigger to respective gene and phenotypic abnormality in development and mortality Target genes Ø Elongation factor 1a (EF-1a): 325bp Ø Ca-channel gene: 152bp Ø CHS1: 510bp Ø Bursicon: 550bp Ø V-ATPase 4

5 3/8/11 Why mealybug (Phenacoccus solenopsis) Virus vector (PVX) Sucking insect Convenience of rearing High fecundity Multiple host plants Available round the year ter (A) (B) (C) Bioassay on Mealybug RNAi Calcium channel Transgenic-PVX inoculated Nicotiana benthamiana Experimental plants in insect rearing cages ter (A) (B) (C) Bioassay on Mealybug RNAi Elongation factor1alpha Bursicon RNAi in P. solenopsis: CHS1 RNAi in P. solenopsis: 1. Control 1. Control 2, 3. Fed on transgenic PVXinoculated plants 2, 3. Fed on transgenic PVXinoculated plants Figure: Suppression of CHS1 gene as a result of RNAi L- Ladder, 1,2 CHS1-Treated (RNAi); 3,4 CHS1- Control 5-8 B-Actin 5

6 Take home message Insects genes can be suppressed by expressing insect-specific dsrna in transgenic plants Thank you RNAi protect against insect damage Why important? Alternative to toxins More acceptable than toxin proteins in food crops 6