AgoArmet and AgoC002: key effector proteins in cotton aphids host adaptation

Abstract

Aphids are insects that feed on phloem and introduce effector proteins into plant cells through saliva. These effector proteins are key in regulating host plant defense and enhancing aphid host adaptation. We identified these salivary proteins in the cotton aphids genome and named them AgoArmet and AgoC002. Multiple sequence alignment, protein structure analysis, and phylogenetic analysis of these proteins with related proteins from other insects showed that AgoArmet and Armet of Aphis craccivora have high sequence identity (97%) and belong to the same evolutionary branch and that AgoC002 shares the highest sequence identity (80%) and closest evolutionary relationship with C002 of Aphis glyceins. Expression profiling of AgoArmet and AgoC002 showed that they were most highly expressed in cotton aphids during the adult-3d period. Cotton aphids transferred to zucchini leaves resulted in a significant increase in the expression of AgoArmet and AgoC002 within 48h. To investigate the functions of AgoArmet and AgoC002, we decreased the expression of these genes in cotton using virus-induced gene silencing (VIGS), which ultimately led to a 38% and 26% decrease in cotton aphids fecundity, respectively. Moreover, the reduction in AgoC002 expression resulted in a significant (24%) reduction in body weight. Taken together, our findings demonstrate that AgoArmet and AgoC002 are key effector proteins involved in cotton aphids feeding and host adaptation.

Keywords: Armet; C002; VIGS; aphid; plant defense; salivary protein.

Figure 1

Phylogenetic analysis of Armet and C002 proteins from different insect orders. The names of proteins in the phylogenetic tree consist of the accession number of the sequence, gene name, and Latin species name. The red box indicates the evolutionary branch of Armet in Aphididae, and the green box indicates the evolutionary branch of C002 in Aphididae. Blue, yellow, and purple modules indicate the evolutionary branches of Armet in Diptera, Lepidoptera and Hymenoptera, respectively.

Figure 2

Amino acid sequence analysis of AgoArmet and AgoC002. Multiple sequence alignments and phylogenetic trees of Armet (A) and C002 (B) proteins from (A) gossypii and other aphids. The red line indicates the signal peptide, the red boxes indicate the cysteine sites, and the black box indicates the endoplasmic reticulum retention motif. The tertiary structures of the AgoArmet (C) and AgoC002 (D) proteins were constructed using SWISS-MODEL.

Figure 3

AgoArmet and AgoC002 expression profiles. We examined the expression of AgoArmet (A) and AgoC002 (B) in the head, thorax, and abdomen at different developmental periods. Different letters indicate significant differences according to Tukey’s HSD test (P < 0.05).

Figure 4

Effect of cotton aphids transfer to zucchini leaves on the expression of (A) AgoArmet and (B) AgoC002. Student's t-test was used for significance analysis (ns, not significant, *0.01 < p < 0.05, **0.001 < p < 0.01, and ***p < 0.001).

Figure 5

Detection of target gene silencing efficiency in VIGS plants. (A) Experimental groups and treatments. Cotton plants were transfected with vectors for knockdown of AgoArmet (B) and AgoC002 (C) expression using virus-induced gene silencing (VIGS). Armet and C002 represent cotton plants transfected with target gene disruptors, P1+P2 represent cotton plants transfected with empty vectors (pTRV1+ pTRV2), and CK represents wild-type cotton plants. Different letters indicate significant differences according to Tukey’s HSD test (P < 0.05).

Figure 6

Functional analysis of AgoArmet and AgoC002. Changes in the number of cotton aphids offspring (A) and body weight (B) were recorded after VIGS knockdown of AgoArmet and AgoC002 expression. Armet and C002 represent cotton plants transfected with target gene disruptors, P1+P2 represent cotton plants transfected with empty vectors (pTRV1+ pTRV2), and CK represents wild-type cotton plants. Different letters indicate significant differences according to Tukey’s HSD test (P < 0.05).