An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters
Fig 5
PsCRN108 interacts with Arabidopsis HSP gene promoters and HSE’s.
(A) Enrichment of AtHSP gene promoters as determined by ChIP assays. Transgenic Arabidopsis plants expressing GFP or GFP:PsCRN108 infected with P. capsici at 10 hpi (I) or without (N) infection were used for ChIP assays. Specific primers were designed for four fragments (S1-4 are described in S7 Fig) in the five selected Arabidopsis HSP promoters (AtHSP20, AT1G53540; AtHSP70, AT3G12580; AtHSP90, AT5G52640; AtHSP101, AT1G74310; and AT5G56010, another HSP90 gene used as a negative control). qPCR analysis was performed on immunoprecipitated DNA using anti-GFP antibodies. Values for the ChIP samples were firstly normalized to the input control and then divided by the no-antibody control to obtain the fold enrichment values. Values are the means ± SEM of three independent biological replicates (**, P<0.01; *, P<0.05 compared with GFP without infection; Dunnett’s test). (B) Yeast one-hybrid (Y1H) assay of PsCRN108 with the AtHSP90.1 promoter region and HSE. A 500-bp (-1 to -500) fragment of the AtHSP90.1 promoter, a 19 bp HSE (CCAGAAGCTTCCAGAAGCC), or a 19 bp HSE mutant (HSEm, CCAtAAGCTTaCAtAAGCC) were integrated into the genome of yeast upstream of the AUR1-C gene to produce yeast bait strains. The lowest concentrations of Aureobasidin A (AbA) that limited the growth of yeast bait strains were determined before transformation with the pGAD plasmid (AtHSP90.1–500, 200 ng ml-1; HSE, 500 ng ml-1; HSEm, 500 ng ml-1) and were used to assess the pGAD transformants. Yeast growth on selective medium (-Leu +AbA) was recorded on day 3 as an indicator of protein—DNA interactions. pGAD:AtHsfA1a served as the positive control and pGAD as the negative control.