A Re-examination of the Role ofAUXIN RESPONSE FACTOR 8in Developmental Abnormalities Caused by the P1/HC-Pro Viral Suppressor of RNA Silencing
Authors: Mlotshwa Sizolwenkosi , Pruss Gail J. , Macarthur John L. , Reed Jason W. , Vance Vicki 
Published: June 2016

AbstractPlant viral suppressors of RNA silencing induce developmental defects similar to those caused by mutations in genes involved in the microRNA (miRNA) pathway. These abnormalities were originally thought to reflect a pleiotropic impact of silencing suppressors on miRNA control of plant development. However, subsequent work with the P1/HC-Pro potyviral suppressor of silencing showed that global impairment of the miRNA pathway was not responsible for the phenotypical anomalies. More recently, developmental defects caused by aP1/HC-Protransgene under control of the 35S promoter were attributed to moderate upregulation ofAUXIN RESPONSE FACTOR 8 (ARF8), a target of miR167. The key piece of evidence in that work was that the developmental defects in the 35S-pro:P1/HC-ProtransgenicArabidopsiswere greatly alleviated in the F1 progeny of a cross with plants carrying thearf8-6mutation.Arf8-6is a SALK line T-DNA insertion mutant, a class of mutations prone to inducing transcriptional silencing of transgenes expressed from the 35S promoter. Here we report a re-investigation of the role ofARF8in P1/HC-Pro-mediated developmental defects. We characterized the progeny of a cross between our 35S-pro:P1/HC-Pro transgenicArabidopsisline and the samearf8-6T-DNA insertion mutant used in the earlier study. The T-DNA mutation had little effect in the F1 generation, but almost allarf8-6/P1/HC-Proprogeny had lost the P1/HC-Pro phenotype in the F2 generation. However, this loss of phenotype was not correlated with the number of functional copies of theARF8gene. Instead, it reflected transcriptional silencing of the 35S-pro:P1/HC-Protransgene, as evidenced by a pronounced decrease in P1/HC-Pro mRNA2accompanied by the appearance of 35S promoter siRNAs. Furthermore,arf8-8, an independent loss-of-function point mutation, had no detectable effects on P1/HC-Pro phenotype in either the F1 or F2 generations. Together these data argue against the reported role of increasedARF8expression in mediating developmental defects inP1/HC-Protransgenic plants.Author SummaryRNA silencing is an important antiviral defense in plants that uses small RNA molecules to target the invading RNA. Plant viruses, however, have countered with proteins that suppress RNA silencing, and one of the best-studied plant viral suppressors of silencing is P1/HC-Pro. When the genetic model plantArabidopsis thalianais bioengineered to express P1/HC-Pro, the resulting plants display distinct developmental abnormalities. These abnormalities are thought to arise because P1/HC-Pro also interferes with the arm of RNA silencing that uses small RNAs called microRNAs (miRNAs) to regulate expression of the plant's own genes. Earlier work, however, showed that interference with all miRNAs in general could not be responsible for these developmental defects. More recently, it was reported that enhanced expression of a single miRNA-controlled gene,AUXIN RESPONSE FACTOR 8 (ARF8), underlies the developmental defects caused by P1/HC-Pro. However, using the sameARF8mutation as that report, as well as a second, independentARF8loss-of function mutation, we now show that mis-regulation ofARF8is not responsible for those defects. One or a few key miRNA-controlled factors might, in fact, underlie the developmental defects caused by P1/HC-Pro; however, our results show thatARF8is not one of the key factors.