The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development.
The helper component-proteinase (HC-Pro) protein of potyviruses is a suppressor of gene silencing and has been shown to elicit plant developmental-defect-like symptoms. In Zucchini yellow mosaic virus (ZYMV), a mutation in the highly conserved FR180NK box of HC-Pro to FI180NK causes attenuation of these symptoms. At 5 days postinoculation and before symptoms appear, virus accumulation, HC-Pro protein levels, and viral short interfering RNA (siRNA) levels are similar for the severe (FRNK) and attenuated (FINK) strains. At this stage, ZYMV(FRNK) caused greater accumulation of most microRNAs (miRNAs), and especially of their complementary miRNA "passenger" strands (miRNA*s), in systemically infected leaves than the attenuated ZYMV(FINK) did. HC-Pro(FRNK) specifically bound artificial siRNA and miRNA/miRNA* duplexes with a much higher affinity than the mutated HC-Pro(FINK). Further analysis of the mutant and wild-type HC-Pro proteins revealed that suppressor activity of the ZYMV HC(FINK) mutant was not diminished. However, the FINK mutation caused a loss of HC-Pro suppressor function in other potyviruses. Replacement of the second positively charged amino acid in the ZYMV FRNK box to result in FRNA also caused symptom attenuation and reduced small RNA duplex-binding affinity without loss of suppressor activity. Our data suggest that the highly conserved FRNK box in the HC-Pro of potyviruses is a probable point of contact with siRNA and miRNA duplexes. The interaction of the FRNK box with populations of miRNAs directly influences their accumulation levels and regulatory functions, resulting in symptom development.
Shiboleth
Yoel Moshe
YM
Department of Plant Pathology, Agricultural Research Organization, the Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel.
Haronsky
Elina
E
Leibman
Diana
D
Arazi
Tzahi
T
Wassenegger
Michael
M
Whitham
Steven A
SA
Gaba
Victor
V
Gal-On
Amit
A
D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D028464:Cucurbita; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D035683:MicroRNAs / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010935:Plant Diseases / Q000276:immunology / Q000821:virology*; D017800:Potyvirus / Q000502:physiology*; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D035683:MicroRNAs; D034741:RNA, Small Interfering; D012367:RNA, Viral; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C072775:HC-Pro protein, potyvirus