SgR1, Encoding a Leucine-Rich Repeat Containing Receptor-like Protein, Is a Major Aphid (Schizaphis graminum) Resistance Gene in Sorghum

Abstract

Greenbug, Schizaphis graminum, is one of the important cereal aphid pests of sorghum in the United States and other parts of the world. Sorghum bicolor variety PI 607900 carries the Schizaphis graminum resistance (SgR1) gene that underlies plant resistance to greenbug biotype I (GBI). Now, the SgR1 has been determined as the major gene conferring greenbug resistance based on the strong association of its presence with the resistance phenotype in sorghum. In this study, we have successfully isolated the SgR1 gene using a map-based cloning approach, and subsequent molecular characterization revealed it encodes a leucine-rich repeat containing receptor-like protein (LRR-RLP). According to DNA sequence analysis, the SgR1 gene are conserved among GBI-resistance sorghum accessions but are variable within susceptible lines. Furthermore, an InDel (-965 nt) at its promoter region and a single-nucleotide polymorphism (SNP, 592 nt) in the CDS of the SgR1 were detected and they are well conserved within resistant genotypes. When the SgR1 gene was cloned and transferred into Arabidopsis plants, the SgR1 was activated in the transgenic Arabidopsis plants in response to attack by green peach aphids according to the results of the histochemical assay, and GUS activity was detected in situ in spots around the vasculature of the leaf where the phloem is located, suggesting its biological function in those transgenic Arabidopsis plants. Overall, this study confirms that the SgR1 gene coding for an LRR-RLP is the major resistance gene to greenbug, a destructive pest in sorghum and wheat. This represents the first greenbug resistance gene cloned so far and indicates that the simple-inherited GBI resistance gene can be used for sorghum improvement with genetic resistance to GBI via molecular breeding or cross-based conventional breeding technologies.

Keywords: LRR-RLP; aphid; gene cloning; host plant resistance; insect resistance; sorghum.

Figure 1

High-resolution genetic map of the SgR1 on chromosome 9 of S. bicolor. (a) Genetic linkage map after QTL validation showing the position of the SgR1 flanked by GSR286 and GSR298 and narrowed into GSR342 and GSR336 with additional markers. (b) Plants (lines 376 and 112) with critical crossovers flanking the SgR1. The SgR1 was delimited into a region flanked with dCAPS marker SP49 and marker GSR383. The white and red circles indicate the point where the chromosomes broke and then reattached to another chromosome. (c) A single putative gene in the candidate region predicted by sorghum reference genome (v.2.1), FGENESH and Gramene. Solid arrows represent exon and transcription orientation. (d) Sequence comparison of the SgR1 promoter regions and open reading frame between the resistant parent PI607900 and the susceptible BTx623. White bar represents promoter region. Solid arrow represents exon and transcription orientation. Nucleotide variants and their respective position in the SgR1 from each genotype were indicated above or below the gene. Amino acid change was placed on corresponding side of nucleotide-variants.

Figure 2

Predicted amino acid sequence of the SgR1 gene product. The deduced protein domains are indicated as a signal peptide and transmembrane domain (bold), unknown function, LRRNT, unknown function, LRR domains with leucine (L in red), and a COOH-terminal tail. Nucleotide variations resulting in the respective amino acid changes between parental lines are underlined.

Figure 3

(a) SNP analyses and multiple nucleotide sequence alignment of the SgR1 alleles in different lines, in which the conserved bases are highlighted in orange or light green and key variations were framed by a red rectangle and same nucleotide variants at a single position are highlighted with the same highlight color. (b) Shows the partially deduced proteins with variations. (c) An unrooted phylogenetic tree constructed with the SgR1 and other RLPs proteins using MEGA software (version version 4.0.) (https://www.megasoftware.net/, accessed on 19 November 2024), and the numbers at nodes represent bootstrap values per 1000 replicates as determined by NJ method. Protein accession numbers used in the tree construction are as follows: Cf-4 (CAA05268, Solanum habrochaites), Cf-2 (AAC15779, Solanum pimpinellifolium), Cf-9 (A55173, Lycopersicon esculentum), HcrVf2 (CAC40826, Malus floribunda), LepR3 (AGC13587, Brassica napus) Cf-5 (AAC78591, Solanum lycopersicum), Rlm2 (KM097068, Brassica napus). (d) Shows sequence variations in the promoter regions of various genetic lines. Different colors in the figure indicate those in different subgroups.

Figure 4

Multi-sequence alignment of the SgR1 with other published RLP R proteins. Purple arrows indicate the amino acid variants in the SgR1 proteins. Amino acids in a column that have similar properties are highlighted with the same colors.

Figure 5

Analysis of the SgR1 in response to GBI infestation using qRT-PCR, showing the relative expression of the gene in three genotypes using the 2^−ΔΔCt method. (a), relative expression of the SgR1 gene in three lines (BTx623, PI607900, and 347-1 at 3, 6, and 9 dpi in response to GBI infestation vs. none-infested control; and (b) relative expression of the SgR1 gene in three lines (BTx623, PI607900, and 347-1 at 3, 6, and 9 dpi compared to 0 dpi. No data means no expression data because seedlings in susceptible line were severely damaged at 9 dpi. Error bars represent standard error among replicates (n = 3) and the asterisks represent statistically significant changes between the controls and aphid infested samples as determined using Student’s t-test, * p <  0.05, ** p <  0.01, *** p <  0.001 while the bars without asterisk are non-significant (p > 0.05).

Figure 6

SgR1p::GUS expression in GPA-infested and non-infested Arabidopsis leaves. The leaf with colonized GPA aphids at 2 dpi (a) and showing histochemical staining (see blue spots in the yellow square) for GUS activity in the leaf (b). GUS activity was not detectable on the non-infested leaves (c,d). Blue colorations showing SgR1p::GUS expression at both vasculatures (e) and other tissues (f) of infested leaves. Scaled bars 200 µm.