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. 2010 Jul 20;107(29):13177-82.
doi: 10.1073/pnas.0910943107. Epub 2010 Jul 6.

Pseudomonas syringae hijacks plant stress chaperone machinery for virulence

Joanna Jelenska  1 , Jodocus A van Hal, Jean T Greenberg
Affiliations

Pseudomonas syringae hijacks plant stress chaperone machinery for virulence

Joanna Jelenska et al. Proc Natl Acad Sci U S A. .

Abstract

Plant heat shock protein Hsp70 is the major target of HopI1, a virulence effector of pathogenic Pseudomonas syringae. Hsp70 is essential for the virulence function of HopI1. HopI1 directly binds Hsp70 through its C-terminal J domain and stimulates Hsp70 ATP hydrolysis activity in vitro. In plants, HopI1 forms large complexes in association with Hsp70 and induces and recruits cytosolic Hsp70 to chloroplasts, the site of HopI1 localization. Deletion of a central P/Q-rich repeat region disrupts HopI1 virulence but not Hsp70 interactions or association with chloroplasts. Thus, HopI1 must not only bind Hsp70 through its J domain, but likely actively affects Hsp70 activity and/or specificity. At high temperature, HopI1 is dispensable for P. syringae pathogenicity, unless excess Hsp70 is provided. A working hypothesis is that Hsp70 has a defense-promoting activity(s) that HopI1 or high temperature can subvert. Enhanced susceptibility of Hsp70-depleted plants to nonpathogenic strains of P. syringae supports a defense-promoting role for Hsp70.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
HopI1Pma is a virulence factor on many crops. Arabidopsis, tobacco, and peas were infiltrated with bacteria (OD600 = 0.0003), and bacterial growth was quantified 3 days after inoculation (dpi). Tomato and mustard family plants were sprayed with bacteria (OD600 = 0.005), and bacterial growth was quantified 7 dpi. Deletion of hopI1 resulted in reduced bacterial growth (*P < 0.05). Gray bars, PmaΔhopI1 strain; white bars, PmaES4326 strain. Growth experiments were repeated two or more times with similar results.
Fig. 2.
Fig. 2.
HopI1 specifically interacts with Hsp70. (A) The J domain of HopI1 specifically interacts with full-length Arabidopsis Hsp70s in vitro. (Upper) Arabidopsis Hsp70 fused to GST and J domain of HopI1 fused to His6 were expressed in E. coli (SDS/PAGE gel stained with Coomassie blue is shown; GST proteins are from one gel and His-proteins from a different gel). cp(C), C-terminal part of cpHsp70-2 (Hsp70-7, amino acids 413–718); cyt, cytosolic Hsp70-1; ER, ER Bip2 (Hsp70-11); GST, GST control; J, J domain of HopI1Pma (amino acids 334–432, 12 kDa with a His tag); c, HopX2 control (40 kDa). (Lower) GST-pull down. Recombinant GST-Hsp70s and GST control were immobilized on glutathione-agarose and incubated with an extract from E. coli expressing the J domain of HopI1 with a His tag (J) or control His6-HopX2 (c). Eluted proteins were separated by SDS/PAGE and detected with Coomassie stain (GST proteins) and with His6 antibody. HopI1 J domain interacted with different full length Hsp70s but not with the C-terminal half (C) of cpHsp70-2 (the N-terminal part of Hsp70s is necessary for the interaction with J proteins; ref. 15). Strong signal in GST control is cross-reaction with GST (27 kDa), not visible in GST-Hsp70s, because GST alone was purified in a higher amount because of differences in solubility. Pulled-down J protein was not detected by Coomassie stain. Signals for all samples are from one exposure of one continuous membrane/gel. (B) HopI1 specifically interacts with Hsp70 from pea chloroplasts. Recombinant His6-HopI1Psy was immobilized on Ni2+-NTA and incubated with pea chloroplast extract (cp). Eluted proteins were separated by SDS/PAGE and detected with Coomassie stain (His6 proteins) and antibody that specifically recognizes chloroplast cpHsp70. Control is His6-HopX2. Signals for all samples are from one exposure of one continuous membrane except recombinant protein input (E. coli extracts), which are from different gel than pull-down samples. (C) HopI1 stimulates ATPase activity of Hsp70 in vitro. (Left) White bars, human Hsp70 (h70); gray bars, GST-AtHsp70-1 (cyt70); -, no J protein; Hlj1, yeast J domain-GST (positive control); HopI, His6-HopI1Psy; QAA, His6-HPD/QAA HopI1Psy mutant. ATP hydrolysis was measured by using 0.3 μM Hsp70 and 0.5 μM J protein, in duplicates. Average of Hsp70 ATPase activity after 2.5 h assayed in three experiments (using two different recombinant protein preparations) is shown with SEs. Recombinant proteins expressed and purified from E. coli are shown on Right [HopI1, QAA, and AtHsp70-1 (cyt70) are from one gel and Hlj1 and human Hsp70 (h70) from another gel].
Fig. 3.
Fig. 3.
HopI1 forms complexes with Hsp70 in planta and induces Hsp70 levels. (A) Hsp70 is a major interactor of HopI1 in planta. Proteins were immunoprecipitated with anti-HA matrix from control (WT) and HopI1Pma-HA-expressing Arabidopsis, separated by SDS/PAGE and stained with Coomassie blue. Experiment was repeated with plants infected with PmaΔhopI1 with the same results. Strong bands were identified by LC-MS/MS as Hsp70 isoforms and HopI1. *, not specific (antibody). (B) J domain and HPD loop are necessary for HopI1 interaction with Hsp70. P/Q repeats of HopI1 are dispensable for this interaction. (Left) Western blots with HA antibody show that chloroplast-enriched fractions from transgenic Arabidopsis were also enriched in HopI1Pma-HA variants (I, HopI1; Δr, Δrepeats; ΔJ, ΔJ domain; Q, HPD/QAA mutant; v, vector control plants) comparing with total extracts. Plants expressing HopI1 and Δrepeats variants had elevated levels of cytHsp70, especially associated with chloroplasts (quantification is shown in Fig. S4B), whereas levels of chloroplast Hsp70 isoforms were not changed. Coomassie-stained membrane (Rubisco) shows similar loading. The same membrane was incubated with cytosolic Hsp70 monoclonal antibody and later with cpHsp70 polyclonal antibody and stained with Coomassie blue; HA signals are from another membrane with the same samples. Signals for all extract samples are from one exposure of one continuous membrane. (Right) Proteins from chloroplast-enriched extracts were immunoprecipitated with anti-HA matrix (due to uneven accumulation of HopI1-HA variants, twice more plant extract was used for IP of QAA and ΔJ than for HopI1 and Δr), separated by SDS/PAGE and stained with Coomassie blue or detected by HA antibody or cytosolic Hsp70 antibody (in separate gels/membranes). cytHsp70 precipitated with HopI1 and Δrepeats. cpHsp70 was not detected with cpHsp70 antibody in IP. Signals for all IP samples are from one exposure of one continuous membrane. IPs and immunoanalyses were repeated at least twice each with transgenic Arabidopsis plants and different HopI1 variants transiently expressed in N. benthamiana (Fig. S2), with the same results. LC-MS/MS analysis was done for two independent IPs from Arabidopsis total extracts and one from chloroplasts. (C) HopI1 and Hsp70 form high molecular mass complexes in planta. Blue-native gel of protein leaf extracts from WT and HopI1-HA (I)-expressing Arabidopsis shows HopI1 in 240–480 kDa complexes (Top). In plants expressing HopI1, Hsp70 is recruited to such high molecular mass complexes (300–350 kDa), larger than Hsp70 complexes in WT plants. The same membrane was incubated with HA antibody, and later with cytosolic Hsp70 antibody and stained with Coomassie blue. Two-dimensional gels of the same samples in Fig. S3 show that signals are from proteins of correct sizes. Large cytHsp70 complexes of similar size as in HopI1-expressing plants also formed in plants infected with Pma 1 d after infiltration at OD600 = 0.01 or spraying at OD600 = 0.1 (Middle). Levels of Pma and PmaΔhopI1 (ΔI) bacteria were similar 1 dpi (P > 0.3; Bottom). –, uninfected plants. Signals for infiltrated plants are from one exposure of one membrane and for sprayed plants from another membrane. (D) Interaction with Hsp70 depends on HopI1’s HPD loop. Recombinant His6-HopI1Psy (I) and HPD/QAA mutant of HopI1Psy (Q) were immobilized on Ni2+-NTA and incubated with Arabidopsis protein extract. Extract from E. coli transformed with empty vector was a control (v). Eluted proteins were separated by SDS/PAGE and detected with Coomassie stain (His6 proteins) and cytHsp70 antibody. Inputs are E. coli and plant extracts. Signals for all pull-down samples are from one exposure of one continuous membrane and input samples are from another membrane. Pull-down experiments were done twice with similar results. (E) P/Q repeats are necessary for virulence function of HopI1. Growth of PmaΔhopI1 strain in planta (infiltrated at OD600 = 0.0003; 3 dpi) was rescued in HopI1-expressing, but not HopI1Δrepeats-expressing Arabidopsis (protein accumulation in transgenic plants is shown in B). *P < 0.05. Growth experiments were repeated at least twice with each of two independent HopI1Δrepeats transgenic lines, giving similar results. (F) HopI1 induces and recruits Hsp70 to chloroplasts during infection. Cytosolic Hsp70 was induced by infection and recruited to chloroplasts to a greater extent when Pma harbored the HopI1 effector. Chloroplast-resident cpHsp70 levels were unaffected by infection. The same membrane was incubated with cytosolic Hsp70 monoclonal antibody and later with cpHsp70 polyclonal antibody and stained with Coomassie blue. Pma, PmaES4326 extracts showing that the cytHsp70 antibody does not recognize bacterial proteins; cp, chloroplast extracts; ΔI, PmaΔhopI1; Pma, PmaES4326; –, uninfected plants. Arabidopsis was sprayed with bacteria at OD600 = 0.1 or infiltrated at OD600 = 0.01 (shown) and Hsp70 levels were examined by Western blot analysis 1 dpi, when levels of both bacteria strains were similar (C). The average amount of cytosolic Hsp70 associated with chloroplasts was 2.2 times higher and total cytosolic Hsp70 1.5 times higher in plants infected with WT Pma than PmaΔhopI1 (Fig. S4B). At least six independent samples in two or more experiments were evaluated. Signals for all samples are from one exposure of one continuous membrane.
Fig. 4.
Fig. 4.
Hsp70 is necessary for HopI1 virulence function. (A) Compared with WT Col, the difference in growth of ΔhopI1 and WT Pma strains was significantly smaller on Arabidopsis with decreased amounts of Hsp70-1 (on average ≈10% of the difference on WT Col in five experiments), cpHsp70-1 (≈2%) and (to lower degree) Hsp70-2 (≈20%). a, growth of ΔhopI1 strain was higher on hsp70-1, hsp70-2, and cphsp70-1 mutants than on WT Arabidopsis (P < 0.005); *, growth of Pma strain was higher than ΔhopI1 on Col, hsp70-2, hsp70-3, Hsp70 OE, and cphsp70-2 plants (P < 0.05). In some experiments (3 of 5), growth of the Pma strain was slightly lower on hsp70-1 mutant than on WT Arabidopsis (P < 0.05) and slightly higher (*) than the growth of ΔhopI1 (P < 0.05). 70-1, 70-2, 70-3, 70 OE,hsp70 mutants, and overexpressing plants; cp-1 and cp-2, chloroplast cphsp70 mutants. Plants were spray inoculated at OD600 = 0.01, and bacterial growth was assayed 3 and 5 dpi (shown). (B) Silencing Hsp70-1 in N. benthamiana complemented growth defect of ΔhopI1 strain. Plants were infected with PVX-vector (PVX-v), PVX-NbHsp70-1 silencing construct (70-1), or mock treated with buffer (mock). Eighteen days later, upper leaves were infiltrated with P. syringae at OD600 = 0.0003 and bacterial growth was assayed 3 dpi. a, growth of ΔhopI1 strain was higher on hsp70-1 silenced N. benthamiana than on control (P < 0.05); *P < 0.002. Western blots in A and B show Hsp70 proteins detected with cytosolic Hsp70 antibody in mutant and silenced plants. Samples shown are from the same membrane exposure. Expression of chloroplast Hsp70 in cphsp70 mutants was reported (23). (C) At 30 °C, ΔhopI1 strain grew as well as WT Pma on Col and hsp70 mutants (P > 0.05). At this temperature, HopI1 was needed for virulence only on plants overexpressing Hsp70 (*P < 0.05). Plants initially grown at 20 °C were transferred and kept at 30 °C after infection. (D) Acute heat shock (35 min at 50 °C; HS) before infection at 20 °C also abolished the growth defect of PmaΔhopI1, as in plants kept at 30 °C after infection (P > 0.07). On Arabidopsis acclimated to 30 °C 1 d before infection (acclim.), HopI1 was needed for full Pma virulence (*P < 0.01). The growth experiments were repeated two (HS, acclimation) or more times (all other experiments) with similar results.
Fig. 5.
Fig. 5.
Hsp70-1 has a role in basal resistance. (A) Type III secretion-deficient strains (C−, hrcC−) of Pma, PtoDC3000 (Pto), and PsyB728a (Psy) grew to higher levels on hsp70-1-silenced N. benthamiana than control PVX-treated plants; the growth of Pma with AvrRpt2 (AvrR2) was not affected. Bacteria were infiltrated at OD600 = 0.01 and growth was measured 3 dpi. (B) Pma hrcC−grew more on Arabidopsis hsp70-1 mutant than on WT plants and cphsp70-1 mutant when bacteria where infiltrated (at OD600 = 0.01) or sprayed (at OD600 = 0.1) and on HopI1-expressing Arabidopsis infected by spraying. *P < 0.05. The growth was measured 5 dpi. a and b indicate that the growth of bacteria was higher on hsp70-1 than HopI1 plants (P < 0.05). These experiments were repeated twice with similar results.
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