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A sorghum genome-wide association study (GWAS) identifies a WRKY transcription factor as a candidate gene underlying sugarcane aphid (Melanaphis sacchari) resistance

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A WRKY transcription factor identified through forward genetics is associated with sorghum resistance to the sugarcane aphid and through heterologous expression reduces aphid populations in multiple plant species.

Abstract

Crop plant resistance to insect pests is based on genetically encoded traits which often display variability across diverse germplasm. In a comparatively recent event, a predominant sugarcane aphid (SCA: Melanaphis sacchari) biotype has become a significant agronomic pest of grain sorghum (Sorghum bicolor). To uncover candidate genes underlying SCA resistance, we used a forward genetics approach combining the genetic diversity present in the Sorghum Association Panel (SAP) and the Bioenergy Association Panel (BAP) for a genome-wide association study, employing an established SCA damage rating. One major association was found on Chromosome 9 within the WRKY transcription factor 86 (SbWRKY86). Transcripts encoding SbWRKY86 were previously identified as upregulated in SCA-resistant germplasm and the syntenic ortholog in maize accumulates following Rhopalosiphum maidis infestation. Analyses of SbWRKY86 transcripts displayed patterns of increased SCA-elicited accumulation in additional SCA-resistant sorghum lines. Heterologous expression of SbWRKY86 in both tobacco (Nicotiana benthamiana) and Arabidopsis resulted in reduced population growth of green peach aphid (Myzus persicae). Comparative RNA-Seq analyses of Arabidopsis lines expressing 35S:SbWRKY86-YFP identified changes in expression for a small network of genes associated with carbon–nitrogen metabolism and callose deposition, both contributing factors to defense against aphids. As a test of altered plant responses, 35S:SbWRKY86-YFP Arabidopsis lines were activated using the flagellin epitope elicitor, flg22, and displayed significant increases in callose deposition. Our findings indicate that both heterologous and increased native expression of the transcription factor SbWRKY86 contributes to reduced aphid levels in diverse plant models.

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Data availability

All data generated or analyzed during this study are included in this published article. Raw reads for RNA-Seq data generated in this study have been deposited to the NCBI Sequence Read Archive (SRA) and are available under the BioProject accession numbers PRJNA786556 and PRJNA786558.

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Acknowledgements

We thank Dr. Georg Jander (Boyce Thompson Institute, Ithaca, NY) for sharing his colony of tobacco-adapted Myzus persicae. Funding for this research was supported in part by the Department of Energy (DOE) Joint Genome Institute (JGI) Community Science Program (CSP) Project # 503420 (to A.H. and E.A.S) and the USDA-ARS NACA #58-6048-7-037, which was part of the research project entitled “Areawide Pest Management of Invasive Sugarcane Aphid in Grain Sorghum (62048-21220-018-00D).” Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

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Correspondence to Alisa Huffaker.

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Communicated by Anastasios Melis.

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Supplementary Information

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425_2021_3814_MOESM1_ESM.tif

Supplementary file1 Quantile–quantile (Q-Q) plots of GWAS analyses. The Q-Q plots were generated by comparing the observed and estimated –log10(P) values produced in the GWAS for sugarcane aphid (SCA) damage ratings in sorghum. The 95% confidence interval is shaded for each plot. Plots were generated using rMVP (Memory-efficient, Visualization-enhanced, and Parallel-accelerated R package) from the results of a GLM(PCA), b MLM(PCA+K) and c FarmCPU algorithm models (TIF 93 KB)

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Supplementary file 2 Comparison of sorghum SCA resistance-associated locus with corresponding maize syntenic genome region using CoGe. SbWRKY86 (Sobic.009G238200, denoted with a star) and ZmWRKY106 (Zm00001d009619) were selected as the reference genes for generating the GEvo analysis. For the sorghum genomic analysis, the sequence was expanded by 176 kb to the left and 180 kb to the right to include approximately 20 genes on each side. For the maize genomic analysis, the sequence was expanded by 712 kb to the left and 498 kb to the right. High-scoring Sequence Pairs shorter than 500 bp were excluded (TIF 208 KB)

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Supplementary file3 Phylogeny tree of all sorghum, maize and Arabidopsis WRKYs as predicted by PlantTFDB. Protein sequences of primary transcripts for Arabidopsis, sorghum and maize were obtained from phytozome and annotated using PlantTFDB to identify all WRKY domain-containing genes. The phylogenetic tree was constructed using IQ-TREE and visualized using FigTree. Symbols were manually assigned based on available WRKY family annotations from Arabidopsis (arabidopsis.org), maize (maizegdb.org) and sorghum (Baillo et al. 2020; https://doi.org/10.1371/journal.pone.0236651). The sorghum SbWRKY86 was colored in blue and maize ZmWRKY106 in green. The complete list of WRKY genes, gene abbreviations and predicted protein sequences used are provided in Supplementary Table 6 (TIF 615 KB)

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Supplementary file 4 Analysis of W-box motifs in the promoters of genes significantly upregulated in Arabidopsis lines expressing 35S::SbWRKY86-YFP. a. A logo file representing the W-box motif predicted by PlantTFDB to be bound by SbWRKY86/AtWRKY30. This motif was found to be statistically enriched in the promoters of genes significantly upregulated in Arabidopsis lines expressing 35S::SbWRKY86-YFP as calculated by the SEA function in MEME suite. Promoters were defined as the region 1 kb upstream from the transcription start site (TSS), and enrichment was determined by comparison to W-box content of all other Arabidopsis gene promoters as a control. b Enumeration of W-box motif content across the promoters of the SbWRKY86-responsive genes as determined through FIMO (from MEME suite) scanning (significance threshold P < 0.001). These identified W-box motifs are summarized by plotting the c number of W-box motifs in each 100 bp interval of the promoters of SbWRKY86-responsive genes and d the distribution of the number of motifs identified in the promoters of SbWRKY86-responsive genes (TIF 410 KB)

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Supplementary file 5 Callose deposition after SCA infestation of an SCA-resistant vs. -susceptible sorghum line. a Quantification of the average callose deposits in leaves of the SCA-susceptible line PI 597950 versus the SCA-resistant line PI 656043 3 days post-infestation with 10 wingless adult SCA. Callose was visualized via aniline blue staining. b Enumeration of SCA 3 days post-infestation on the resistant vs. susceptible plants used for callose deposition assays. For all n = 4, ± SEM, with statistical differences between genotypes analyzed by a pairwise Student’s t-test (TIF 27 KB)

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Supplementary file 6 Proposed model for enhanced resistance to aphid attack conferred through heterologous expression of SbWRKY86. When wild type Arabidopsis plants are infested with M. persicae, endogenous immune activation results in defense responses, including callose deposition, that does not sufficiently limit aphid proliferation. In plants expressing 35S::SbWRKY86-YFP, endogenous immune activation is augmented by increased expression of genes associated with regulation of C/N metabolism, immune responses and callose production. As one mechanism contributing to resistance, plants expressing SbWRKY86-YFP display increased callose deposition, which could lead to more effective restriction of aphid feeding and proliferation (TIF 891 KB)

Supplementary file7 (XLSX 71798 KB)

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Poosapati, S., Poretsky, E., Dressano, K. et al. A sorghum genome-wide association study (GWAS) identifies a WRKY transcription factor as a candidate gene underlying sugarcane aphid (Melanaphis sacchari) resistance. Planta 255, 37 (2022). https://doi.org/10.1007/s00425-021-03814-x

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