A helitron-induced RabGDIα variant causes quantitative recessive resistance to maize rough dwarf disease

Maize rough dwarf disease (MRDD), caused by various species of the genus Fijivirus, threatens maize production worldwide. We previously identified a quantitative locus qMrdd1 conferring recessive resistance to one causal species, rice black-streaked dwarf virus (RBSDV). Here, we show that Rab GDP dissociation inhibitor alpha (RabGDIα) is the host susceptibility factor for RBSDV. The viral P7-1 protein binds tightly to the exon-10 and C-terminal regions of RabGDIα to recruit it for viral infection. Insertion of a helitron transposon into RabGDIα intron 10 creates alternative splicing to replace the wild-type exon 10 with a helitron-derived exon 10. The resultant splicing variant RabGDIα-hel has difficulty being recruited by P7-1, thus leading to quantitative recessive resistance to MRDD. All naturally occurring resistance alleles may have arisen from a recent single helitron insertion event. These resistance alleles are valuable to improve maize resistance to MRDD and potentially to engineer RBSDV resistance in other crops.

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Sample sizes for all fine-mappings or transgenic validations are listed in the figures.In the fine-mapping of resistance QTL qMrdd1, we adopted a sequential mapping strategy based on recombinant-derived progeny testing (Yang, et al., 2012. A sequential quantitative trait locus finemapping strategy using recombinant-derived progeny. J. Integrative Plant Biol. 54, 228-237). Since qMrdd1 could explain~30% of the phenotypic variation, theoretically, 20 individuals could significantly (P < 0.05) detect the difference in disease severity index (DSI) between individuals with and without qMrdd1 in the same progeny. In the current research, we normally used 25-135 individuals in each progeny, and could authentically detect the significant difference in DSI between the two genotypes.In the transgenic validation of candidate gene, we adopted a paired student's t-test or a student t-test to test for significant difference in DSI values between transgenic and non-transgenic plants. A range of 17 to 167 individuals were tested for each genotype, and this proved to be sufficient to test for the genetic contribution of each candidate gene on disease control.For RT-qPCR, each leaf tissue had three samples, and each sample was harvested from five plants ( only upper leaves). RNA expression for each sample was tested in triplicate. No.
In the transgenic verification of candidate gene, we set up repetition in regard with both locations and viruliferous planthoppers. The plants from the same transgenic event was grown in Taian (Shandong province) or Beijing for artificial inoculation with different sources of viruliferous planthoppers. In a split-luciferase complementation assay, more than five leaves (five replicates) were used for each assay. In other protein interaction assays, like pull down, co-immunoprecipitation and competition assay, at least three replicates were set up for each experiment.
In the fine-mapping of qMrdd1, a recombinant-derived progeny was grown in the same plot, therefore, all individuals, with or without qMrdd1, were randomly distributed in the same experimental plot. We then calculated the DSI values for the genotypes with and without qMrdd1 and tested for their significant difference in DSI values. This could maximally reduce the influence of environmental conditions on two genotypes in disease resistance, please refer to Yang, et al. (Yang, et al., 2012. A sequential quantitative trait locus fine-mapping strategy using recombinant-derived progeny. J. Integrative Plant Biol. 54, 228-237). For the transgenic verification of the candidate gene, we adopted different strategy for different transgenic events. In validation of the susceptible ZmGDI gene, the segregating progeny was grown in the same plot after artificial inoculation, each individual was scored for its disease resistance and investigated for its genotype. The DSI values, calculated for both transgenic and non-transgenic plants, were used to test for significant difference in disease resistance between ransgenic and non-transgenic subgroups. In validation of the resistance ZmGDI gene, the pure transgenic and non-transgenic lines were firstly inoculated with viruliferous planthoppers and then randomly grown in the experimental plots. The same procedure was adopted for the transgenic events with RNAi constructs.
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October 2018
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