Importance of HopI1 and its J domain for P. syringae virulence. Data represent the means of 8 samples with standard errors.
(A) Schematic structure of HopI1PmaES4326 showing different protein regions.
(B) The ΔhopI1 strain with the empty vector pCKTR (black bar) grew significantly less than PmaES4326 with empty vector (white bar) on many A. thaliana accessions and two tobacco species (*P<0.05). This phenotype was complemented by hopI1PmaES4326 (JJ19, dark-gray bar) or hopI1PmaES4326 with a point mutation in J domain (hopI1H387Q, JJ77, light-gray bar) expressed from the native promoter and integrated into the chromosome.
(C) Epitope-tagged versions of HopI1 were expressed in the ΔhopI1 PmaES4326 strain from native promoter in conditions that promote effector protein production. Proteins were detected with anti-HA antibody by SDS-PAGE and Western blot analysis: (1) JJ19=hopI1-HA-Myc-His, (2) pCKTR, (3) JJ78= hopI1-HA, (4) JJ77=hopI1H/Q-HA-Myc-His, (5) JJ207=hopI1HPD/QAA-HA-Myc-His. Lines 1–3 are from one gel.
(D) The ΔhopI1 strain carrying an HA-tagged hopI1 allele (gray bar) from any of several P. syringae strains expressed from the nptII promoter grew significantly more on Columbia (Col) plants than ΔhopI1 strain carrying the control vector pME6012 (black bar) (*P<0.0001): (1) hopI1PmaES4326; JJ78; (2) hopI1PtoDC3000, JJ152; (3) hopI1PsyB728a; JJ151; (4) hopI1PsyCit7, JJ153; (5) hopI1Psy61, JJ154. White bar, wild type PmaES4326 strain with empty vector.
(E) Left panel: The J-domain triple mutant HPD/QAA of HopI1 integrated into the chromosome under native promoter (JJ207) did not complement the growth defect of ΔhopI1 strain in Col and Nossen (No). Strains with different letters showed significant differences in their growth (P<0.001, Fisher’s protected least significant difference measure (PLSD), a post hoc, multiple t-test). ΔI,ΔhopI1 PmaES4326 strain; v, vector pCKTR; I, full-length HopI1 (JJ19); I(QAA), HPD/QAA mutant of HopI1 (JJ207). Right panel: representative Col leaves from the infection with the Δ carrying vector (v), full length HopI1 (hopI1) or the QAA mutant shown in the left panel.
(F) The growth of the ΔhopI1 strain was not rescued by hopI1Δ (JJ193) or hopIΔJ (JJ194) integrated into the chromosome under native promoter. However, the ΔhopI1 strain containing hopI1Δ showed a trend of being partially complemented compared to the vector control (§P<0.056, Fisher’s PLSD). Strains with different letters show significant differences in growth (P<0.02, Fisher’s PLSD).