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. 2015 May 20;10(5):e0126161. doi: 10.1371/journal.pone.0126161

Fig 5. Characterization of Y398 on NSs self-interaction and protein stability.

Fig 5

(A) Self-interaction analysis of NSs and mutants protein. Each co-transformed yeast cells grown on SD medium lacking Histidine (His), Leucine (Leu) and Tryptophan (Trp) without or with 2 mM of 3-AT. AD: activation domain, BD: binding domain. (B) Western blotting for detection of NSs protein expressed in yeast cells which were transformed with wild type NSs or Y398A construct. (C) Western blotting for detection of individual point-mutated NSs constructs, including wild type Y, mutated A, D, E, S, T, and F residues at the aa position 398 of NSs protein, co-infiltrated with empty vector (EV) or HC-Pro (HC-Pro) construct at 4 days post-agroinfiltration (dpa). Anti-NSs MAb was used for detecting NSs protein. Coomassie blue stained RuBisCO proteins were used as loading controls. (D) The RNA silencing suppression function of mutated NSs, Y398A, Y398D and Y398F, analyzed at 4 dpa. The relative proportions of Agrobacterium was 1:0.5:1 for GFP:GFi:NSs. (E) The self-interaction of mutated NSs, Y398A, Y398D and Y398F were examined by yeast two hybrid analysis. (F) Symptoms on squash plants after inoculation of individual ZYMV recombinants carrying Y398A or Y398F at 14 days post-infection.