Creation of a Dominant-Negative Mutant of Autophagy Protein ATG4B in Nicotiana benthamiana and its Effects on Degradation of Proteins Targeted by P0

HARBESON 17

RNA silencing, a plant immune mechanism, eliminates viral RNA to prevent viral infection. For viruses to successfully infect their host plants, RNA silencing must be overcome by a virulence factor. P0, a protein from the Turnip yellows virus (TuYV) and Potato leaf roll virus (PLRV), suppresses RNA silencing via degradation of key host enzymes called Argonaute proteins (AGOs). Degradation of these host proteins has been linked to both the proteasome-mediated degradation pathway and autophagic pathway. ATG4 is a family of C54 cysteine proteases that modulate autophagy. Overexpression of an inactive dominant-negative mutant of ATG4B with cysteine-74 in the catalytic site substituted with alanine (C74A) was shown to inhibit autophagy in human cells; this has yet to be demonstrated in plants. We hypothesize that introduction of the same cysteine to alanine mutation in ATG4 from Nicotiana benthamiana (NbATG4) would create a dominant-negative mutant that would inhibit the plant autophagic pathway. Reverse transcription-polymerase chain reactions were conducted to make cDNA and amplify the NbATG4A and NbATG4B genes. In humans, homologs HsATG4A and HsATG4B show moderate structural similarities; however, comparison of the two clones from N. benthamiana showed 98% similarity. N. benthamiana is an allotetraploid and this data suggests that the clones are two copies of the gene resulting from the allotetraploidy. Comparison of NbATG4B with the ATG4B gene sequences from Arabidopsis thaliana (AtATG4B) and Homo sapiens (HsATG4B) showed that the plant homologs have more extended amino-termini, so the catalytic site cysteine in NbATG4B was at amino acid residue 173 (C173). Oligonucleotide primers were designed to mutate cysteine-173 into an alanine to construct the dominant negative mutant, NbATG4B^C173A. Wild-type and mutant NbATG4B clones will be useful to gain insight into the autophagic function of P0 in Polerovirus-host interactions and would reveal whether there are similarities in ATG4 function between plants and mammals.