Biochemical evidence of epicuticular wax compounds involved in cotton-whitefly interaction

Abstract

Sucking insects require a surface of plants on which the legs and the eggs of insects will adhere and to which insect mouthparts will access. The primary plant protection against insects is their surface property, which hinders the attachment of the insect's legs and eggs. The epicuticular waxes chemistry influences the fine structure of the cuticular surface. In current study, an attempt was made to investigate the variation of chemical compounds in epicuticular waxes of four cotton species that classify them resistant or susceptible i.e., Gossypium abroreum, G. hirsutum, G. arboreum wax deficient mutant (GaWM3) and G. harknessi which were evaluated for their interaction with whitefly and CLCuV transmission. Gossypium hirsutum an insect and CLCuV susceptible cotton variety, was found to have four compounds namely Trichloroacetic acid, hexadecylester, P-xylenolpthalein, 2-cyclopentene-1-ol, 1-phenyl-and Phenol, 2,5-bis [1,1- dimethyl] which could interact with chitin of whitefly while only two compounds in Gossypium arboreum an insect and CLCuV resistant cotton variety could interact with chitin of whitefly. Similarly, GaWM3 and Gossypium harkasnessi were found to have only a single compound. Number of whiteflies found on leaves of G. hirsutum was much higher as compared to other cotton species. Keeping this fact in mind a wax biosynthetic gene CER3, from Arabidopsis thaliana was transformed into G. hirsutum and the plants were evaluated for their resistance against whitefly and CLCuV transmission. In microscopic analysis transgenic plants clearly showed higher amounts of leaf waxes as compared to non-transgenics. The least whitefly population and CLCuV titer of <10,000 units was found in transgenic plants compared to non-transgenic cotton where it was ≈4.5X106 units that confirmed the role of wax in insect interaction and ultimately to CLCuV transmission. This study provides novel insight on wax related compounds involved in cotton-whitefly interaction, which potentially can help in developing more efficient control strategies for this destructive pest.

Fig 1

(A) Interaction of Trichloroacetic acid, hexadecylester with chitin where the central carbon attached to chlorine is highly deficient to the electron and can interact with the electron pair from chitin. (B) Interaction of P-xylenolpthalein with chitin by making hydrogen bonds.

Fig 2

(A) Interaction of hydrogen of 2-cyclopentene-1-ol-phenyl- with oxygen and hydroxyl group present in chitin. (B) Interaction of hydrogen of Phenol,2, 5, bis [1,1-dimethyl] with oxygen and hydroxyl group present in chitin.

Fig 3

(A) Interaction of piperidinone, n-|4-bromo-n-butyl| with chitin of whitefly (B) Interaction of hydrogen of Lanceol, cis- with oxygen and hydroxyl group present in chitin.

Fig 4

A: Germinated seeds, B: Injuring the embryos with sharp blade on shoot apex side, C: Co-cultivation of embryos with agrobacterium, D: Embryos in growth medium, E: plants in rooting and shooting media, F: Plants in soil pots for acclimatization.

Fig 5. Transgene (CER3) amplification through PCR.

Lane 1: 100bp Ladder, Lane 2: positive control, Lane 3,4,5,6 transgenic plants, Lane 7: negative control (non-transgenic cotton).

Fig 6. Measurement of CLCuV titer (β-satellite) in control and transgenic cotton plants (WL1, WL2, WL3, WL4).

Fig 7

(A) Number of whiteflies visits to different genotypes of cotton (B) The trend of whitefly both on transgenic and control cotton (G. hirsutum) plants.

Fig 8

(A): Non-transgenic (control) Gossypium hirustum after wax removal. (B): Gossypium arboreum after wax removal (C): Transgenic Gossypium hirustum showing higher wax removal after treatment with the organic solvent. (D): Transgenic Gossypium hirustum showing higher wax removal after treatment with the organic solvent.