The Mitochondrial Protein RESISTANCE to APHIDS 9 Interacts with S40 to Resist Aphid Infestation by Modulating Reactive Oxygen Species Homeostasis in Maize (Zea mays)

Abstract

As the corn aphid (Rhopalosiphum maidis) poses a major threat to maize (Zea mays) growth, there is much interest in identifying aphid resistance genes. In this study, an aphid-susceptible maize mutant from an ethyl methanesulfonate-mutagenized library is identified that exhibits greater aphid settlement than the wild type. Using the MutMap approach, the causal gene RESISTANCE TO APHIDS 9 (RTA9) is cloned, which encodes a mitochondrion-localized protein from the Domain of Unknown Function 641 family. Overexpressing RTA9 in maize confers significant resistance to aphids without compromising seed yield. It further identifies the senescence regulator S40 as an interactor of RTA9, which negatively regulates the stability of S40. Knockout of S40 enhanced aphid resistance, while its overexpression increased susceptibility. Further analysis demonstrates that the rta9-1 mutant does not exhibit significant enrichment of differentially expressed genes associated with oxidoreductase activity following aphid infestation. By contrast, genes involved in this pathway are significantly enriched in the s40 mutant. Additionally, aphid-induced reactive oxygen species (ROS) levels are markedly lower in rta9-1 than in the wild type but significantly higher in s40. Collectively, the results suggest that the mitochondrial protein RTA9 and its interacting partner S40 regulate resistance to aphid infestation by modulating ROS homeostasis.

Keywords: DUF641; RTA9; S40; aphids (Rhopalosiphum maidis); maize (Zea mays); mitochondrial protein; reactive oxygen species (ROS) homeostasis.

Figure 1

The rta9‐1 mutant is susceptible to aphids. A) Colonization and distribution of aphids on the wild‐type B73 and the rta9‐1 mutant after 21 days of infestation. Scale bar, 1 cm. B) Quantification of aphids growing on B73 and rta9‐1 as shown in (A). C,D) Honeydew secretion assay of aphid feeding. The quantification of the honeydew areas on the filter papers in (C) is shown in (D). E) Survival rates of aphids feeding on B73 or rta9‐1 (^** p < 0.01, ^*** p < 0.001). F) Body weight of each aphid feeding on B73 or rta9‐1. The boxes represent the interquartile range, with the middle line defining the median. The lines extending from the quartiles of the box are called “whiskers” and show the maximum and minimum values. In panels (B, D–F), at least six independent replicates were performed for each assay, and the data in panels (B, D–E) are presented as means ± standard deviation (SD). The values in (D) were normalized to B73, which was set to 1. Statistical significance of the differences between B73 and rta9‐1 was determined by Student's t‐tests.

Figure 2

MutMap‐based cloning of RTA9 and aphid resistance tests of the rta9‐2 mutant and RTA9‐overexpressing plants. A) Distribution of CG>TA‐type mutations in rta9‐1 across the 10 maize chromosomes, shown as the SNP‐index. The red arrowhead marks the peak over the significance threshold on chromosome 3. B) Diagram of the Zm00001d039444 (RTA9) locus and confirmation of the C‐to‐T mutation in rta9‐1 by Sanger sequencing. The red box shows the codon change from CAG to TAG, a stop codon that is predicted to result in a truncated RTA9 protein. C) Survival rate of aphids feeding on B73 or the independent mutant allele rta9‐2 (^*** p < 0.001). D,E) Honeydew secretion assay. Quantification of the honeydew areas on the filter paper in (E) is presented in (D). F) Relative RTA9 transcript levels in the wild‐type KN5585 and RTA9‐overexpressing (RTA9‐OE) transgenic lines. GAPDH was used as an internal reference for the RT‐qPCR analysis. G) Immunoblot analysis indicating that RTA9‐GFP accumulates in the RTA9‐OE transgenic plants. GAPDH was used as a loading control. H) Colonization and distribution of aphids on the wild‐type KN5585 and RTA9‐OE lines after 21 days of infestation. Scale bar, 1 cm. I,J) Honeydew secretion assay. Quantification of the honeydew areas on the filter paper in (I) is shown in (J). K) Survival rate of aphids feeding on KN5585 or RTA9‐OE lines (^** p < 0.01, ^*** p < 0.001). L,M) Number and body weight of colonizing aphids after 21 days of infestation. In (F), data are shown as means ± SD, n = 3. In (C, D, J–L), the data are shown as means ± SD, n = 6. In (M), the boxes represent the interquartile range, with the middle line defining the median. The lines extending from the quartiles of the box are called “whiskers” and show the maximum and minimum values. Statistical significance was determined using Student's t‐tests (C, D, F, K) or one‐way ANOVA followed by Tukey's test (J–M) (p < 0.05), with different letters indicating significant difference.

Figure 3

RTA9 interacts with S40 in vivo and in vitro. A) Co‐localization of RTA9 and S40 in maize protoplasts. Mitochondria were visualized by staining with MitoTracker Red dye. Nuclei were visualized with the nuclear marker NLS‐mCherry. Free GFP was used as a control for RTA9‐GFP. B) Yeast two‐hybrid assay indicating that RTA9 interacts with S40 in yeast cells. Positive colonies were spotted onto synthetic defined (SD) medium lacking Trp and Leu (SD/−Trp−Leu) and SD medium lacking Trp, Leu, His, and Ade (SD/−Trp−Leu−His−Ade). The T + P53 combination was used as a positive control. C) Firefly luciferase (LUC) complementation imaging assay indicating that RTA9 interacts with S40 in the leaves of N. benthamiana plants. D) Bimolecular fluorescence complementation (BiFC) assay showing that RTA9 interacts with S40 in maize protoplasts. E) Co‐immunoprecipitation (Co‐IP) assay demonstrating the interaction between RTA9‐GFP and S40‐FLAG in vivo. The RTA9‐GFP transgenic line was crossed with the S40‐FLAG transgenic line, and the derived F₁ generation plants were used for Co‐IP analysis. F) His protein pull‐down assay revealing that RTA9‐MBP‐His interacts directly with S40‐GST in vitro.

Figure 4

Loss of S40 function confers aphid resistance, while S40 overexpression increases aphid susceptibility in maize. A) Diagram of the Zm00001d003009 (S40) locus and identification of the mutation in s40 by Sanger sequencing. B) Colonization and distribution of aphids after 21 days of infestation of the wild‐type B104 and s40 plants. Scale bar, 1 cm. C,D) Honeydew secretion assay of aphid feeding. Quantification of the honeydew areas on the filter paper in (C) is shown in (D). E) Survival rate of aphids feeding on the wild‐type B104 or s40 mutant (^* p < 0.05, ^** p < 0.01). F) Number of aphids present after 21 days of infestation. (G) Body weight of aphids. H) Relative S40 transcript levels in B104 and S40‐overexpression (S40‐OE) transgenic plants. GAPDH was used as an internal reference. I) Immunoblot analysis indicating that S40‐FLAG accumulates in S40‐OE transgenic plants. GAPDH was used as a loading control. J) Colonization and distribution of aphids after 21 days of infestation. Scale bar, 1 cm. K,L) Honeydew secretion assay of aphid feeding. Quantification of the honeydew areas on the filter paper in (K) is shown in (L). M) Survival rate of aphids (^*** p < 0.001). N,O) Number (N) and body weight (O) of colonizing aphids after 21 days of infestation. In (H), data are shown as means ± SD, n = 3. In (D–F, I–N), data are shown as means ± SD, n = 6. In (G, O), the boxes represent the interquartile range, with the middle line defining the median. The lines extending from the quartiles of the box are called “whiskers” and show the maximum and minimum values. Statistical significance was determined using Student's t‐test (D–H) or one‐way ANOVA followed by Tukey's test (L–O) (p < 0.05), with different letters indicating significant differences.

Figure 5

RTA9 negatively modulates S40 protein stability in vivo and in vitro. A,B) Fluorescence intensity of S40‐GFP in B73 and rta9‐1 protoplasts (A) and quantification of fluorescence levels (B). C,D) Fluorescence intensity of S40‐GFP in KN5585 and RTA9‐OE protoplasts (C) and quantification of fluorescence levels (D). E,F) Cell‐free degradation assay showing that the degradation of S40 is slower in extracts from the wild‐type KN5585 than from those of RTA9‐OE. Representative immunoblots are presented in (E) and the relative protein abundance is shown in (F). S40‐GST was detected using anti‐GST antibodies, and Coomassie Brilliant Blue (CBB) staining was used to ensure equal loading. In (B, D), the boxes represent the interquartile range, with the middle line defining the median. The lines extending from the quartiles of the box are called “whiskers” and show the maximum and minimum values. In (F), data are shown as means ± SD, and significant differences were determined with Student's t‐tests. Experiments in (E, F) were performed at least three times with similar results.

Figure 6

RTA9 and S40 regulate the cellular redox status. A) Venn diagram showing the extent of overlap between the number of differentially expressed genes (DEGs) in the wild‐type B73, rta9‐1, B104, and s40 (control, CK; Tr, aphid infestation). The set of genes highlighted by the dashed line represents those that exhibit common changes in B73 and s40 in response to aphid infestation. B) Gene Ontology (GO) term enrichment analysis of DEGs in the region highlighted by the gray dashed line in (A). C–F) Imaging of ROS levels, detected with H₂DCFDA, in B73 and rta9 (C) and in B104 and s40 (E) under control conditions (CK) and upon aphid infestation (Tr). Quantification of the fluorescence intensity in (C) and (E) is shown in (D) and (F), respectively. G) 3,3′‐diaminobenzidine (DAB) histochemical staining of leaves from B73, rta9‐1, B104, and s40 plants under control conditions or in response to aphid infestation. H) Nitroblue tetrazolium (NBT) histochemical staining of leaves from B73, rta9‐1, B104, and s40 plants under control conditions or in response to aphid infestation. In panels (D, F), at least six individual leaves were stained and photographed. Fluorescence intensity was quantified from 100 randomly selected regions (n = 100). The boxes represent the interquartile range, with the middle line defining the median. The lines extending from the quartiles of the box are called “whiskers” and show the maximum and minimum values. Significant differences were determined using one‐way ANOVA followed by Tukey's test (D and F) (p < 0.05), with different letters indicating significant differences.