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. 2018 Apr 4;9:613. doi: 10.3389/fmicb.2018.00613

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Copyright © 2018 Zhang, Zhao, Li, Xiang, Dong, Zhang, Wang, Li, Yu and Han.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Schematic representation of the BrYV-3a mutants generated and northern blot detection of their systemic infection. (A) Representation of BrYV genomic RNA organization. ORFs 0, 1 and 2 are translated from the gRNA with expression of ORF1 being dependent on leaky scanning and translation of ORF2 via a –1 ribosomal frameshift. ORFs 3a, 3, 4, and 5 are translated from sgRNA1, with expression of ORF3a being ACG initiation, of ORF4 via leaky scanning, and of ORF5 via stop codon readthrough. The position of the ORF3a is highlighted in yellow. Nucleotide mutations (red) were introduced into ORF3a and the substitutions are indicated for each mutant (right). (B,C) RNAs extracted from N. benthamiana upper leaves were submitted to northern blot analyses. The plants infiltrated with the wild-type BrYV or the mutants BrYV^C3405A (BrYV^P18T), BrYV^C3405U (BrYV^P18S), BrYV^C3405G (BrYV^P18A), BrYV^C3406A (BrYV^P18Q), BrYV^C3406U (BrYV^P18L), BrYV^C3406G (BrYV^P18R), BrYV^A3407C, BrYV^A3407U, BrYV^A3407G, BrYV^C3467U, BrYV^A3381G (BrYV^T10A), BrYV-P3aAUG (AUG), BrYV-P3aAGC (AGC), respectively. B, Each sample corresponds to a mixture of leaves from three individual plants. C. Each sample corresponds to an individual plant. (D) Symptoms of N. benthamiana infiltrated leaves by agro-infiltration with BrYV mutants observed 5 dpi. (E) Northern blot analysis of RNAs extracted from N. benthamiana leaves infiltrated with WT or the mutants. (F) Western blot analysis of the accumulation of BrYV CP, MP, and CP-RTD in agroinfiltrated N. benthamiana leaves at 2 dpi. Coomassie brilliant blue (Coom.) staining is shown as a loading control. Mock, negative control; gRNA, genomic RNA; sgRNA, subgenomic RNA, rRNA, loading control of methylene blue stained ribosomal RNA.