Journal: Fundamental Research

Article Title: A key heavy metal-binding protein orchestrates plant resistance against a geminivirus

doi: 10.1016/j.fmre.2024.12.005

Figure Lengend Snippet: NbHMP04 binds to copper ions to stimulate plant immunity. (A) Schematic representation of the conserved domains of NbHMP04 protein. HMA represents heavy metal associated domain; M represents Met; C represents Cys; x represents any amino acid. (B) Copper (Cu) and cadmium (Cd) tolerance of NbHMP04- or NbHMP04 mHMA -transformed yeast cells. Yeast cells transformed with vector pYES2 (as a negative control), pYES2-NbHMP04, or pYES2- NbHMP04 mHMA were grown in SD/-Urea with 2% galactose for 72 h (h), and their OD 600 was adjusted to 1, 0.1, 0.01, 0.001; the diluted cultures were spotted onto medium supplemented with 0 µM,20 µM, 40 µM Cd 2+ , or with 0 µM, 200 µM, 500 µM Cu 2+ . (C) Cu-NTA beads pull down the NbHMP04 protein. Total protein was extracted from GFP-, NbHMP04-GFP-, or NbHMP04 mHMA -GFP-expressing leaves, then incubated with Cu-NTA Beads for 2 h. Anti-GFP antibodies were used to detect the pulled-down proteins. GFP was used as the negative control. (D) Symptoms of wild-type N. benthamiana plants grown in low (0.3 µM) or high (3 µM) Cu 2+ hydroponic solution inoculated with TYLCCNV/TYLCCNB infectious clones or pBinplus (as a negative control) at 6 days post-inoculation (dpi). (E) Quantitative real-time PCR (qPCR) showing accumulation of TYLCCNV CP (left part) and NbPR1 (right part) in systemically infected leaves from (D). Data are presented as means ± SD of three biological replicates. Statistical analyses were performed using the Student's t -test. *, p < 0.05. NbActin was used as the internal reference gene. (F) Symptoms of Solanum lycopersicum plants (upper panel) or nbhmp04 -L6 and nbhmp04 -L8 N. benthamiana plants (lower panel) infected with TYLCCNV/TYLCCNB at 6 dpi. Plants were grown in low or high Cu 2+ hydroponic solution. (G) qPCR showing accumulation of TYLCCNV CP (left panel) and SlPR1 (right panel) in systemically infected leaves from (upper panel of F). Data are presented as means ± SD of three biological replicates. Statistical analyses were performed using the Student's t -test. *, p < 0.05, ***, p < 0.001. SlActin was used as the internal reference gene. (H) qPCR showing accumulation of TYLCCNV CP (left panel) and NbPR1 (right panel) in systemically infected leaves from (lower panel of F). Data are presented as means ± SD of three biological replicates. ns, not significant. NbActin was used as the internal reference gene.

Article Snippet: For the Cu binding assay, special PureCube Cu-NTA MagBeads (cat: 31,501-Cu, Cube Biotech, Rheinland, Germany) were used, and the Co-IP buffer was without EDTA.

Techniques: Transformation Assay, Plasmid Preparation, Negative Control, Expressing, Incubation, Clone Assay, Real-time Polymerase Chain Reaction, Infection