Presentation Title

Toward analysis of selective autophagy receptor, Joka2 in polerovirus P0-induced autophagic degradation

Faculty Mentor

Melanie Sacco

Start Date

23-11-2019 10:45 AM

End Date

23-11-2019 11:30 AM

Location

96

Session

poster 4

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Toward analysis of selective autophagy receptor, Joka2 in polerovirus P0-induced autophagic degradation

Autophagy is the housekeeping process of protein and organelle degradation to recycle nutrients for further use in the cell. This process is also utilized by cells during pathogenic infection. In mammalian cells, the protein receptor p62 guides organelles tagged for degradation with polyubiquitin through the autophagy pathway. In the plant genus Nicotiana, the protein Joka2 is a cargo receptor thought to be the ortholog of mammalian p62. We are investigating how the viral protein P0 from poleroviruses causes the degradation of plant proteins, including the RNA silencing enzyme Argonaute-1 (AGO1) by autophagy. We hypothesize that P0-mediated degradation of target proteins utilizes selective cargo receptors, like Joka2, in AGO1 degradation. The Joka2 cDNA sequence was PCR-amplified from N. benthamiana cDNA with primers incorporating restriction enzyme cut sites and ligated into pGEM-T. A Joka2 cDNA clone verified by DNA sequencing was digested with SpeI and BamHI and ligated into XbaI and BamHI double-digested binary expression vector pBIN61. The final expression clone fused to a hemagglutinin (HA) epitope tag was sent for sequencing to confirm the validity of the construct. This construct can be tested for interaction with AGO1 in the presence of P0 to determine if Joka2 is responsible for AGO1 targeting to the autophagic vesicles.

Keywords: Autophagy, protein degradation, plant virus

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Nov 23rd, 10:45 AM Nov 23rd, 11:30 AM

Toward analysis of selective autophagy receptor, Joka2 in polerovirus P0-induced autophagic degradation

96

Toward analysis of selective autophagy receptor, Joka2 in polerovirus P0-induced autophagic degradation

Autophagy is the housekeeping process of protein and organelle degradation to recycle nutrients for further use in the cell. This process is also utilized by cells during pathogenic infection. In mammalian cells, the protein receptor p62 guides organelles tagged for degradation with polyubiquitin through the autophagy pathway. In the plant genus Nicotiana, the protein Joka2 is a cargo receptor thought to be the ortholog of mammalian p62. We are investigating how the viral protein P0 from poleroviruses causes the degradation of plant proteins, including the RNA silencing enzyme Argonaute-1 (AGO1) by autophagy. We hypothesize that P0-mediated degradation of target proteins utilizes selective cargo receptors, like Joka2, in AGO1 degradation. The Joka2 cDNA sequence was PCR-amplified from N. benthamiana cDNA with primers incorporating restriction enzyme cut sites and ligated into pGEM-T. A Joka2 cDNA clone verified by DNA sequencing was digested with SpeI and BamHI and ligated into XbaI and BamHI double-digested binary expression vector pBIN61. The final expression clone fused to a hemagglutinin (HA) epitope tag was sent for sequencing to confirm the validity of the construct. This construct can be tested for interaction with AGO1 in the presence of P0 to determine if Joka2 is responsible for AGO1 targeting to the autophagic vesicles.

Keywords: Autophagy, protein degradation, plant virus