Auxin Binding Protein 1 Reinforces Resistance to Sugarcane Mosaic Virus in Maize.

Sugarcane mosaic virus (SCMV) causes severe viral diseases in maize worldwide (Fuchs and Gruntzig, 1995Fuchs E. Gruntzig M. Influence of sugarcane mosaic-virus (Scmv) and maize-dwarf mosaic-virus (Mdmv) on the growth and yield of 2 maize varieties.J. Plant Dis. Protect. 1995; 102: 44-50Google Scholar), resulting in significant losses in grain and forage yield in susceptible cultivars of maize and related crops. The most promising solution is to cultivate resistant varieties, which contribute to sustainable crop production. Two epistatically interacting major SCMV resistance loci (Scmv1 and Scmv2) are required to confer complete resistance against SCMV in the resistant near-isogenic line F7RR/RR (the letters left of the slash refer to the genotype at Scmv2 on chromosome 3 and those on the right refer to the genotype at Scmv1 on chromosome 6, with R indicating a resistance allele and S a susceptibility allele) (Xing et al., 2006Xing Y. Ingvardsen C. Salomon R. Lübberstedt T. Analysis of sugarcane mosaic virus resistance in maize in an isogenic dihybrid crossing scheme and implications for breeding potyvirus-resistant maize hybrids.Genome. 2006; 49: 1274-1282Crossref PubMed Google Scholar). Scmv2 adds a second layer of resistance to the immediate response mediated by Scmv1. Scmv1 has recently been identified to encode ZmTrxh, which acts as a molecular chaperone to suppress viral RNA accumulation in cytoplasm without eliciting a salicylic acid- or jasmonic acid-mediated defense response (Liu et al., 2017Liu Q. Liu H. Gong Y. Tao Y. Jiang L. Zuo W. Yang Q. Ye J. Lai J. Wu J. et al.An atypical thioredoxin imparts early resistance to sugarcane mosaic virus in maize.Mol. Plant. 2017; 10: 483-497Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The quantitative nature of SCMV resistance, causing escapes and incomplete penetrance (Xing et al., 2006Xing Y. Ingvardsen C. Salomon R. Lübberstedt T. Analysis of sugarcane mosaic virus resistance in maize in an isogenic dihybrid crossing scheme and implications for breeding potyvirus-resistant maize hybrids.Genome. 2006; 49: 1274-1282Crossref PubMed Google Scholar), impedes the functional validation of Scmv2. By genotyping all recombinants from our previous study (Ingvardsen et al., 2010Ingvardsen C. Xing Y. Frei U. Lübberstedt T. Genetic and physical fine mapping of Scmv2, a potyvirus resistance gene in maize.Theor. Appl. Genet. 2010; 120: 1621-1634Crossref PubMed Scopus (29) Google Scholar), we narrowed Scmv2 down to a 250 kb region on chromosome 3 between markers 184B1 and 212FG08, covered by overlapped B73 (c0023O09, B0654c18, and c0281K07) and FAP1360A (46H09, 223J01, and 009E08) BAC clones (Figure 1A). Five candidate genes were predicted, encoding auxin binding protein 1 (ABP1), glutathione synthase (GSS), and three unknown/hypothetical proteins (Figure 1A). All five candidate genes were sequenced for both resistant FAP1360A and susceptible F7 parental lines (Figure 1B). Non-synonymous variants between FAP1360A and F7 alleles were exclusively found for ZmABP1 and ZmGSS genes. We used a transgenic approach to evaluate the contribution of candidate genes to SCMV resistance. T0 RNAi transgenic plants, either carrying pMCG1005-ABP1 or pMCG1005-GSS constructs (Supplemental Figure 1A), were first crossed with F7RR/RR and then backcrossed to F7RR/RR (Xing et al., 2006Xing Y. Ingvardsen C. Salomon R. Lübberstedt T. Analysis of sugarcane mosaic virus resistance in maize in an isogenic dihybrid crossing scheme and implications for breeding potyvirus-resistant maize hybrids.Genome. 2006; 49: 1274-1282Crossref PubMed Google Scholar), and BC1 plants homozygous for the Scmv2 resistance allele were selected for comparison. No plants carrying pMCG1005-GSS construct were symptomatic during 4 weeks of observation. From four transgenic events, a total of 10 BC1 plants harboring the pMCG1005-ABP1 construct showed obvious SCMV symptoms within 4 weeks post inoculation (wpi) (Figure 1C) with a significantly (P = 0.05) lower ZmABP1 expression level (Supplemental Figure 1B) and a larger amount of SCMV accumulated in symptomatic transgenic plants compared with non-transgenic siblings determined by ELISAenzyme-linked immunosorbent assay (ELISA) (Supplemental Figure 1C). Thus, we focused on ZmABP1 in subsequent experiments. The ZmABP1 alleles of FAP1360A and F7 were designated ABP1FAP and ABP1F7, respectively. The complementation construct (pTF-ABP1FAP) including native full-length geneABP1FAP (Figure 1D) was introduced into the maize transformable genotype HiII, and T0 progeny were crossed with near-isogenic line F7SS/RR and backcrossed to F7SS/RR (Xing et al., 2006Xing Y. Ingvardsen C. Salomon R. Lübberstedt T. Analysis of sugarcane mosaic virus resistance in maize in an isogenic dihybrid crossing scheme and implications for breeding potyvirus-resistant maize hybrids.Genome. 2006; 49: 1274-1282Crossref PubMed Google Scholar). For transgenic event of Aux7-1, 96% BC1 plants lacking the pTF-ABP1FAP construct were susceptible, in contrast to 45% symptomatic transgenic plants carrying pTF-ABP1FAP (Figure 1E). For Aux 29-1, 92% of plants lacking pTF-ABP1FAP were susceptible, in contrast to 55% carrying pTF-ABP1FAP (Figure 1E). Exogenous ABP1FAP significantly (P = 0.01) increased SCMV resistance, which was associated with higher ZmABP1 expression levels (Figure 1F). To identify causal variants responsible for resistance, we characterized ZmABP1 genomic sequences from resistant inbreds FAP1360A, Pa405, D32, D21, 1145, and susceptible lines F7, D145, and Mo17. No consistent differences were found between resistance and susceptibility alleles. Major differences between FAP1360A and F7 are five SNPs and a 524 bp insertion in FAP1360A (Supplemental Figure 2). FAP1360A and F7 alleles share the same cDNA sequence (603 bp) from RACE (rapid amplification of cDNA ends), except for two non-synonymous SNPs (A18C and A85G) in the first exon, and three synonymous SNPs (G333C, T483A, and A492G) in the third and fourth exons. The 524 bp insertion was also found in Pa405, which is distantly related to FAP1360A. A deletion construct (pTF-dABP1FAP) was created by deleting the 524 bp region from the pTF-ABP1FAP construct (Figure 1D), and validated in transgenic plants as complementation construct. Of 80% plants lacking pTF-dABP1FAP were symptomatic, in contrast to 18% of plants carrying pTF-dABP1FAP from transgenic event Mid9-1, as well as for transgenic event Mid15-4 (Figure 1G). Increased resistance of plants harboring pTF-dABP1FAP suggest that the 524 bp insertion/deletion has no or negligible impact on SCMV resistance. ZmABP1 expression level again was significantly (P = 0.01) associated with the disease resistance (Figure 1H). Increased ZmABP1 expression was observed at 14 days post inoculation (dpi) for both F7 and F7RR/RR plants compared with mock treatment, and it was 1.7-2.2 fold higher for inoculated F7RR/RR compared with inoculated F7 at later time points (Figure 1I). In contrast, no late response was found in the expression of other positional candidate genes (Supplemental Figure 3). The higher and late SCMV-induced ZmABP1 expression level is consistent with the previous finding that Scmv2 is active at later stages post SCMV infection (Xia et al., 1999Xia X.C. Melchinger A.E. Kuntze L. Lübberstedt T. Quantitative trait loci mapping of resistance to sugarcane mosaic virus in maize.Phytopathology. 1999; 89: 660-667Crossref PubMed Scopus (77) Google Scholar, Xing et al., 2006Xing Y. Ingvardsen C. Salomon R. Lübberstedt T. Analysis of sugarcane mosaic virus resistance in maize in an isogenic dihybrid crossing scheme and implications for breeding potyvirus-resistant maize hybrids.Genome. 2006; 49: 1274-1282Crossref PubMed Google Scholar). We characterized allelic ZmABP1 promoter activity for FAP1360A and F7 using maize protoplast transient expression assays. Compared with the 1711 bp ABP1F7 promoter, the corresponding ABP1FAP promoter segment led to 2-fold and thus substantial and significant increase (P = 0.01) of LUC activity (Figure 1J). We observed a slightly but not significantly increased LUC activity for the 1281 bp promoter of ABP1FAP compared with ABP1F7. However, no significant difference was found between the respective 430 bp promoters (Figure 1J). These results indicate that the 430 bp ABP1FAP promoter segment between −1282 bp and −1711 bp contains a functional cis-regulatory element, promoting luciferase activity and by inference, ZmABP1 expression in vivo, thereby promoting SCMV resistance. We identified two cis elements in the FAP1360A 430 bp promoter region that are absent in the corresponding region of F7: a BoxII motif, and three Sp1 motifs arranged in tandem. Both are light responsive, which might be required for increased ZmABP1 expression levels in response to light, resulting in light-dependent virus resistance (Chandra-Shekara et al., 2006Chandra-Shekara A.C. Gupte M. Navarre D. Raina S. Raina R. Klessig D. Kachroo P. Light-dependent hypersensitive response and resistance signaling against turnip crinkle virus in Arabidopsis.Plant J. 2006; 45: 320-334Crossref PubMed Scopus (135) Google Scholar). The endoplasmic reticulum (ER) is the site for potyvirus 6K2 protein-induced vesicle assembly and viral replication (Restrepohartwig and Carrington, 1994Restrepohartwig M.A. Carrington J.C. The tobacco etch Potyvirus 6-kilodalton protein is membrane-associated and involved in viral replication.J. Virol. 1994; 68: 2388-2397PubMed Google Scholar). ZmABP1 encoded by either ABP1FAP or ABP1F7 colocalized with ER- (CD3-959) and plasma membrane (PM) (CD3-1007)-specific markers, revealing the presence of ZmABP1 at both ER and PM (Figure 1K; Supplemental Figures 4–6). These findings suggest no detectable effect of two polymorphic amino acids of ZmABP1 on its subcellular localization in either resistant or susceptible genotypes. Viral RNA colocalized with ER, where the virus replication complex (VRC) is initiated and viral replication takes place. We evaluated the effect of ZmABP1 alleles on SCMV replication using a transient assay based on maize protoplasts in vivo. SCMV replication was not impaired by increasing ZmABP1 expression (Supplemental Figure 7). This suggests that ER-localized ZmABP1 has no effect on SCMV replication and is not the cause of SCMV resistance. We next screened for ZmABP1-interacting proteins by yeast two-hybrid assays using F7RR/RR derived ABP1 as bait, and obtained ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbCS), which showed the strongest interaction with ZmABP1 (Supplemental Figure 8). Recently, NtRbCS was reported to be the host target of potyvirus P3 protein and Tobamovirus multifunctional movement protein for virion replication (Danci et al., 2009Danci O. Ziegler A. Torrance L. Gasemi S. Danci M. Potyviridae family - short review.J. Hortic. For. Biotechnol. 2009; 13: 410-420Google Scholar) and systemic movement (Zhao et al., 2013Zhao J.P. Liu Q. Zhang H.L. Jia Q. Hong Y.G. Liu Y.L. The rubisco small subunit is involved in tobamovirus movement and Tm-22-mediated extreme resistance.Plant Physiol. 2013; 161: 374-383Crossref PubMed Scopus (67) Google Scholar). Transiently expressed fluorescence was detected in Nicotiana benthamiana lines co-infiltrated with pABP1-YFPC and pRbCS-YFPN through a bimolecular fluorescence complementation (BiFC) assay (Supplemental Figure 9). Co-immunoprecipitation (Co-IP) assays further demonstrated an interaction between ZmABP1 and RbCS (Supplemental Figure 10). ZmRbCS also interacted with ABP1F7 (Supplemental Figure 11). Whether and how the interaction between ZmABP1 and ZmRbCS is associated with SCMV resistance remains unknown; further studies are needed to reveal the ZmABP1-mediated SCMV resistance mechanism. Scmv1 most probably suppresses SCMV replication and slows local spread as the “frontline of defense” probably around 2 wpi (like F7SS/RR) (Xing et al., 2006Xing Y. Ingvardsen C. Salomon R. Lübberstedt T. Analysis of sugarcane mosaic virus resistance in maize in an isogenic dihybrid crossing scheme and implications for breeding potyvirus-resistant maize hybrids.Genome. 2006; 49: 1274-1282Crossref PubMed Google Scholar). When SCMV overcomes this “frontline of defense”, rapid systemic movement follows via sieve elements. RbCS might be recruited for VRC formation by viral proteins and for systemic movement. ZmABP1 expression is greatly stimulated by SCMV infection in resistant genotypes, reinforcing resistance to viruses that escaped from the Scmv1 barrier at later stages. As a consequence, ZmRbCS would neither be available for SCMV genome amplification nor utilized for potyvirus long-distance movements (Danci et al., 2009Danci O. Ziegler A. Torrance L. Gasemi S. Danci M. Potyviridae family - short review.J. Hortic. For. Biotechnol. 2009; 13: 410-420Google Scholar, Zhao et al., 2013Zhao J.P. Liu Q. Zhang H.L. Jia Q. Hong Y.G. Liu Y.L. The rubisco small subunit is involved in tobamovirus movement and Tm-22-mediated extreme resistance.Plant Physiol. 2013; 161: 374-383Crossref PubMed Scopus (67) Google Scholar). Scmv2 also confers resistance to other potyviruses, such as Maize dwarf mosaic virus and Wheat streak mosaic virus (Lübberstedt et al., 2006Lübberstedt T. Ingvardsen C. Melchinger A.E. Xing Y. Salomon R. Redinbaugh M.G. Two chromosome segments confer multiple potyvirus resistance in maize.Plant Breed. 2006; 125: 352-356Crossref Scopus (19) Google Scholar). Our study provides evidence for a crucial role of ZmABP1 in potyvirus resistance and will be beneficial for breeding of potyvirus multiresistant cultivars, adding an unexpected and exciting aspect to its biological functions. Further studies toward understanding the molecular and genetic mechanisms of ZmABP1-mediated SCMV resistance are still needed. This work was supported by RF Baker Center for Plant Breeding at Iowa State University, the Danish Research Council and the Danish International Development Agency (DANIDA), the USAID iAGRI project, and China Postdoctoral Science Foundation (grant no. 2015M580149).