Figure 5.

ACIK1 Is Required for Both Cf-9– and Cf-4–Dependent HRs in N. benthamiana and Tobacco.

(A) VIGS in N. benthamiana using TRV. Seedlings were inoculated with TRV:ACIK1, TRV:SGT1, or control TRV:00. Three weeks after initiation of silencing, one leaf half was infiltrated with Agrobacteria that direct Cf-9 and Avr9 expression. Pictures of two independent leaves (A and B) were taken 3 d after agroinfiltration (top panel).

(B) To analyze NbACIK1 transcript levels, RNA was isolated from the other leaf half of each leaf shown in (A) before and 30 min after infiltration of water (flooding) to induce NbACIK1 expression. NbACIK1 transcript accumulation was analyzed by RT-PCR. Actin was used as an internal control for equal cDNA loading (bottom panel).

(C) RNAi in N. tabacum. pBIN19 constructs containing the NtACIK1/NtACIK1 hairpin (A/A) or containing a nonhairpin control NtACIK1/NtCDPK2 (A/C) were transiently expressed in Cf-9 transgenic tobacco plants. Four days after Agrobacteria were infiltrated, the leaves were challenged with various dilutions of IF(+Avr9), and the development of HR was observed for 3 d.

(D) On parallel leaves, NtACIK1 transcript accumulation was determined as described in (B).