Development and Evaluation of Stable Sugarcane Mosaic Virus Mild Mutants for Cross-Protection Against Infection by Severe Strain.

Sugarcane mosaic virus (SCMV; genus Potyvirus) induces maize dwarf mosaic disease that has caused serious yield losses of maize in China. Cross-protection is one of the efficient strategies to fight against severe virus strains. Although many mild strains have been identified, the spontaneous mutation is one of the challenging problems affecting their application in cross-protection. In this study, we found that the substitution of cysteine (C) at positions 57 or 60 in the zinc finger-like motif of HC-Pro with alanine (A; C57A or C60A) significantly reduced its RNA silencing suppression activity and SCMV virulence. To reduce the risk of mild strains mutating to virulent ones by reverse or complementary mutations, we obtained attenuated SCMV mutants with double-mutations in the zinc finger-like and FRNK motifs of HC-Pro and evaluated their potential application in cross-protection. The results showed that the maize plants infected with FKNK/C60A double-mutant showed symptomless until 95 days post-inoculation and FKNK/C60A cross-protected plants displayed high resistance to severe SCMV strain. This study provides theoretical and material bases for the control of SCMV through cross-protection.

Development and application of a full-length infectious clone of potato virus Y isolate belonging to SYR-I strain.

Potato virus Y (PVY) causes huge damage to potato and tobacco production worldwide. The complete genome sequence of GZ, a PVY isolate (strain SYR-I) from Guizhou province, China, was cloned into the binary vector pCambia0390. Three introns were individually inserted into the P3 and CI ORFs to produce plasmid pCamPVY-GZ. The plasmid could infect plants of Nicotiana benthamiana, N. tabacum via agroinfiltration and plants of pepper and potato by mechanical inoculation. The green fluorescence protein gene of Aequoria victoriae was cloned into the encoding regions between nuclear inclusion body 'b' and coat protein genes in pCamPVY-GZ to produce pCamPVY-GZ-GFP, which could infect plants of N. benthamiana, N. tabacum, potato and tomato, and produce green fluorescence in the systemic leaves of inoculated plants. Mutations were introduced to pCamPVY-GZ to make the lysine (K) 391 and glutamic acid (E)410 of helper component-proteinase to arginine (R) and asparagic acid (E), respectively. Unlike wild type PVY-GZ, the mutant PVY-K391R/E410D could not induce veinal necrosis in N. tabacum plants. With an interval of 14 days, mutant PVY-K391R/E410D could protect N. tabacum plants from the infection of severe PVY strain. The results presented here provide a promising alternate for the prevention of diseases caused by PVY.

Recombination of strain O segments to HCpro-encoding sequence of strain N of Potato virus Y modulates necrosis induced in tobacco and in potatoes carrying resistance genes Ny or Nc.

Hypersensitive resistance (HR) to strains O and C of Potato virus Y (PVY, genus Potyvirus) is conferred by potato genes Ny(tbr) and Nc(tbr), respectively; however, PVY N strains overcome these resistance genes. The viral helper component proteinases (HCpro, 456 amino acids) from PVY(N) and PVY(O) are distinguished by an eight-amino-acid signature sequence, causing HCpro to fold into alternative conformations. Substitution of only two residues (K269R and R270K) of the eight-amino-acid signature in PVY(N) HCpro was needed to convert the three-dimensional (3D) model of PVY(N) HCpro to a PVY(O) -like conformation and render PVY(N) avirulent in the presence of Ny(tbr), whereas four amino acid substitutions were necessary to change PVY(O) HCpro to a PVY(N) -like conformation. Hence, the HCpro conformation rather than other features ascribed to the sequence were essential for recognition by Ny(tbr). The 3D model of PVY(C) HCpro closely resembled PVY(O), but differed from PVY(N) HCpro. HCpro of all strains was structurally similar to ß-catenin. Sixteen PVY(N) 605-based chimeras were inoculated to potato cv. Pentland Crown (Ny(tbr)), King Edward (Nc(tbr)) and Pentland Ivory (Ny(tbr)/Nc(tbr)). Eleven chimeras induced necrotic local lesions and caused no systemic infection, and thus differed from both parental viruses that infected King Edward systemically, and from PVY(N) 605 that infected Pentland Crown and Pentland Ivory systemically. These 11 chimeras triggered both Ny(tbr) and Nc(tbr) and, in addition, six induced veinal necrosis in tobacco. Further, specific amino acid residues were found to have an additive impact on necrosis. These results shed new light on the causes of PVY-related necrotic symptoms in potato.

Genetic determinants of Potato virus Y required to overcome or trigger hypersensitive resistance to PVY strain group O controlled by the gene Ny in potato.

Potato virus Y (PVY) (genus Potyvirus) is the most economically damaging and widely distributed virus in potato. Spread of PVY in the field is controlled by growing resistant cultivars. The dominant potato gene Ny(tbr) for hypersensitive resistance (HR) controls ordinary PVY strains (PVY(O)) but is overcome by PVY(N) strains. Studies with infectious PVY chimeras and mutants indicated that the viral determinants necessary and sufficient to overcome Ny(tbr) reside within the helper component proteinase (HC-Pro) (residues 227 to 327). Specifically, eight residues and the modeled three-dimensional conformation of this HC-Pro region distinguish PVY(N) from PVY(O) strains. According to the model, the conserved IGN and CCCT motifs implicated in potyvirus replication and movement, respectively, are situated in a coiled structure and an α-helix, respectively, within this region in PVY(O); however, their locations are reversed in PVY(N). Two residues (R269 and K270) are crucial for the predicted PVY(O)-specific HC-Pro conformation. Two viral chimeras triggered Ny(tbr) and induced veinal necrosis in tobacco, which is novel for PVY. One chimera belonged to strain group PVY(E). Our results suggest a structure-function relationship in recognition of PVY(O) HC-Pro by Ny(tbr), reveal HC-Pro amino acid signatures specific to PVY(O) and PVY(N), and facilitate identification of PVY strains overcoming Ny(tbr).