Abstract
Rice blast is one of the most serious diseases of rice and a major threat to rice production. Breeding disease-resistant rice is one of the most economical, safe, and effective measures for the control of rice blast. As a complement to traditional crop breeding, the transgenic method can avoid the time-consuming process of crosses and multi-generation selection. In this study, maize (Zea mays) Activator (Ac)/Dissociation (Ds) transposon vectors carrying green fluorescent protein (GFP) and red fluorescent protein (mCherry) genetic markers were used for generating marker-free transgenic rice. Double fluorescent protein-aided counterselection against the presence of T-DNA was performed together with polymerase chain reaction (PCR)-based positive selection for the gene of interest (GOI) to screen marker-free progeny. We cloned an RNAi expression cassette of the rice Pi21 gene that negatively regulates resistance to rice blast as a GOI into the Ds element in the Ac/Ds vector and obtained marker-free T1 rice plants from 13 independent transgenic lines. Marker-free and Ds/GOI-homozygous rice lines were verified by PCR and Southern hybridization analysis to be completely free of transgenic markers and T-DNA sequences. qRT-PCR analysis and rice blast disease inoculation confirmed that the marker-free transgenic rice lines exhibited decreased Pi21 expression levels and increased resistance to rice blast. TAIL-PCR results showed that the Ds (Pi21-RNAi) transgenes in two rice lines were reintegrated in intergenic regions in the rice genome. The Ac/Ds vector with dual fluorescent protein markers offers more reliable screening of marker-free transgenic progeny and can be utilized in the transgenic breeding of rice disease resistance and other agronomic traits.
Highlights
Rice blast caused by the rice blast fungus (Magnaporthe oryzae) is one of the most serious diseases of rice and a major threat to rice production (Deng et al, 2017)
We constructed a double fluorescent protein-expressing Ac/Ds transposon vector in which the Ds element carrying a gene of interest (GOI) is tandemly linked to Ac transposase (AcTPase), hygromycin phosphotransferase (HPT), green fluorescent protein (GFP), and mCherry
T-DNAharboring T1 progeny can be identified by GFP and mCherry fluorescence assays, and non-fluorescent T1 progeny can be screened by polymerase chain reaction (PCR) assays for Ds and the GOI to obtain marker-free transgenic plants
Summary
Rice blast caused by the rice blast fungus (Magnaporthe oryzae) is one of the most serious diseases of rice and a major threat to rice production (Deng et al, 2017). More than 100 genes or loci conferring rice blast resistance have been identified Most of the cloned rice blast resistance genes are classic dominant R genes. R genes exhibit a phenotype of high resistance or immunity to rice blast, they usually confer resistance to a narrow spectrum of rice blast races and their resistance is lost due to the variation in pathogenicity of M. oryzae. The pi gene does not respond much strongly and rapidly to M. oryzae infection as compared with the race-specific R genes such as Pi9 (Qu et al, 2006) and Pigm (Deng et al, 2017), the pi21-mediated slow defense response might lead to the durability of disease resistance and can be utilized in rice breeding
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