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. 2010 Sep;154(1):233-44.
doi: 10.1104/pp.110.159723. Epub 2010 Jul 12.

Plant immunity directly or indirectly restricts the injection of type III effectors by the Pseudomonas syringae type III secretion system

Emerson Crabill  1 , Anna Joe, Anna Block, Jennifer M van Rooyen, James R Alfano
Affiliations

Plant immunity directly or indirectly restricts the injection of type III effectors by the Pseudomonas syringae type III secretion system

Emerson Crabill et al. Plant Physiol. 2010 Sep.

Abstract

Plants perceive microorganisms by recognizing microbial molecules known as pathogen-associated molecular patterns (PAMPs) inducing PAMP-triggered immunity (PTI) or by recognizing pathogen effectors inducing effector-triggered immunity (ETI). The hypersensitive response (HR), a programmed cell death response associated with ETI, is known to be inhibited by PTI. Here, we show that PTI-induced HR inhibition is due to direct or indirect restriction of the type III protein secretion system's (T3SS) ability to inject type III effectors (T3Es). We found that the Pseudomonas syringae T3SS was restricted in its ability to inject a T3E-adenylate cyclase (CyaA) injection reporter into PTI-induced tobacco (Nicotiana tabacum) cells. We confirmed this restriction with a direct injection assay that monitored the in planta processing of the AvrRpt2 T3E. Virulent P. syringae strains were able to overcome a PAMP pretreatment in tobacco or Arabidopsis (Arabidopsis thaliana) and continue to inject a T3E-CyaA reporter into host cells. In contrast, ETI-inducing P. syringae strains were unable to overcome PTI-induced injection restriction. A P. syringae pv tomato DC3000 mutant lacking about one-third of its T3E inventory was less capable of injecting into PTI-induced Arabidopsis plant cells, grew poorly in planta, and did not cause disease symptoms. PTI-induced transgenic Arabidopsis expressing the T3E HopAO1 or HopF2 allowed higher amounts of the T3E-CyaA reporter to be injected into plant cells compared to wild-type plants. Our results show that PTI-induced HR inhibition is due to direct or indirect restriction of T3E injection and that T3Es can relieve this restriction by suppressing PTI.

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Figures

Figure 1.
Figure 1.
PTI induction times and flagellin concentrations that inhibit the P. fluorescens(pHIR11)-dependent HR in tobacco. PTI was induced by infiltration with 3 × 108 cells/mL of P. fluorescens(pLN1965) or various flg21 concentrations (indicated by black dashed lines). After the specified period, an overlapping infiltration of 3 × 108 cells/mL of the HR-inducing strain P. fluorescens(pHIR11) (indicated by white dashed lines) was performed. The presence or absence of HR in the overlapping region (bordered by white and black dashed lines) was evaluated 48 h after P. fluorescens(pHIR11) infiltration. A, Tobacco leaves were infiltrated with P. fluorescens(pHIR11) 0, 2, 4, or 8 h after having been infiltrated with P. fluorescens(pLN1965). B, Tobacco leaves were infiltrated with P. fluorescens(pHIR11) 0, 0.5, 1, or 2 h after having been infiltrated with 1 μm flg21. C, Tobacco leaves were infiltrated with 0, 0.01, 0.1, or 1 μm of flg21 2 h prior to infiltration with P. fluorescens(pHIR11). The fraction to the left of each image indicates the number of times that the HR was inhibited over the total number of times the assay was performed.
Figure 2.
Figure 2.
PTI restricts injection of T3E-CyaA fusion proteins into tobacco cells. PTI was induced by infiltration with 3 × 108 cells/mL P. fluorescens(pLN1965) or 1 μm flg21 at the indicated times prior to an overlapping infiltration of 3 × 108 cells/mL P. fluorescens(pLN1965) expressing AvrPtoB-CyaA or HopU1-CyaA. The level of T3E injection was determined by quantifying cAMP in the overlapping infiltration area 16 h later. cAMP levels in tobacco leaves infiltrated with P. fluorescens(pLN1965 + pavrPtoB-cyaA) pretreated with flg21 (A), P. fluorescens(pLN1965 + phopU1-cyaA) pretreated with flg21 (B), P. fluorescens(pLN1965 + pavrPtoB-cyaA) pretreated with P. fluorescens(pLN1965) (C), or P. fluorescens(pLN1965 + phopU1-cyaA) pretreated with P. fluorescens(pLN1965) (D). Reduced cAMP levels in PTI-induced plant cells show that PTI restricts T3E injection. Standard error bars are shown, and each experiment was repeated three times with similar results.
Figure 3.
Figure 3.
The ability of DC3000 to elicit an HR and inject T3E-CyaA fusions in tobacco is inhibited by PTI. A, PTI was induced in tobacco leaves by infiltration with 3 × 108 cells/mL of the DC3000 hrcC mutant 0, 2, 4, or 8 h prior to an overlapping infiltration with 2 × 107 cells/mL of the wild-type DC3000 and scored for HR inhibition. The fraction to the left of each image indicates the number of times that the HR was inhibited over the total number of times the assay was performed. B, PTI was induced in tobacco leaves by infiltration of 1 μm flg21 0, 0.5, 1, or 2 h prior to infiltration with 2 × 107 cells/mL DC3000 and scored for HR inhibition. C, The level of injection was determined by measuring cAMP in tobacco 7 h after infiltration with 3 × 108 cells/mL of DC3000(phopU1-cyaA) in plants pretreated with hrcC or flg21 at the times indicated. D, AvrRpt2-HA is cleaved only when present inside plant cells. PTI was induced in tobacco leaves with a 1 μm flg21 treatment prior to infiltration of DC3000 containing a construct that encodes AvrRpt2-HA. In a water (mock) treatment control cleavage of AvrRpt2-HA can be detected with anti-HA antibodies, but no or reduced amounts of cleaved AvrRpt2-HA can be detected in PTI-induced tobacco tissue. Molecular mass markers in kilodaltons are indicated at the left. PTI induction inhibited the HR and the ability of DC3000 to inject HopU1-CyaA or AvrRpt2-HA into tobacco cells. Each experiment was repeated at least three times with similar results. Standard error bars are shown when appropriate.
Figure 4.
Figure 4.
PTI-induced injection restriction occurs in tested P. syringae nonhost interactions but not in compatible interactions. PTI was induced with 3 × 108 cells/mL of the hrcC mutant (left panels) or 1 μm flg21 (right panels). At the times indicated, 3 × 108 cells/mL of bacterial strains expressing HopU1-CyaA were infiltrated into an overlapping area, and cAMP amounts were determined. The following bacteria/plant combinations were used to assess injection capability: A, P. fluorescens(pLN1965 + phopU1-cyaA)/Arabidopsis Col-0; B, DC3000(phopU1-cyaA)/Arabidopsis Col-0, a compatible interaction; C, P. syringae pv tabaci 11258(phopU1-cyaA)/N. tabacum cv Xanthi, a compatible interaction; and D, P. syringae pv tabaci 11258(phopU1-cyaA)/Arabidopsis Col-0, a nonhost interaction. PTI induction severely restricted the ability of bacterial strains in nonhost plants to inject the HopU1-CyaA fusion into plant cells based on the reduced levels of cAMP. However, bacterial strains in host plants were capable of relatively high levels of HopU1-CyaA injection irrespective of PTI induction. Each experiment was repeated at least three times with similar results, and se bars are shown.
Figure 5.
Figure 5.
A DC3000 polyeffector mutant loses its ability to inject T3Es into PTI-induced Arabidopsis. A, Infiltrations of 3 × 108 cells/mL of wild-type DC3000, DC3000 EEL mutant (UNL155), DC3000 effector pathogenicity island (EPai) mutants (UNL158, UNL159, and UNL184), and a DC3000 quadruple mutant (UNL227) each expressing HopU1-CyaA were infiltrated into Arabidopsis after a 0- or 2-h pretreatment with 1 μm flg21, and the production of cAMP was determined. DC3000 single mutants were differentially affected but retained their ability to inject the HopU1-CyaA fusion; however, the DC3000 quadruple mutant was restricted in its T3E injection after PTI induction based on low cAMP levels. B, The virulence of wild-type DC3000, the DC3000 hrcC mutant, the DC3000 single EPai mutants, and the DC3000 quadruple mutant were compared by spray inoculating them at 2 × 107 cells/mL onto untreated Arabidopsis. Virulence was assessed by disease symptoms at 4 d postinoculation (B) and enumeration of bacteria at 0 and 4 d postinfiltration (C). Letters “a” to “d” are statistically different (P < 0.05), and se bars are shown. These data indicate that the quadruple mutant loses the ability to inject T3Es in PTI-induced Arabidopsis, cannot produce disease symptoms, and grows poorly in plant tissue.
Figure 6.
Figure 6.
PTI-induced injection restriction occurs in Arabidopsis when DC3000 carries the avirulence gene avrRpm1. PTI was induced with 1 μm flg21. At the times indicated, 3 × 108 cells/mL of DC3000 strains expressing HopU1-CyaA and with or without AvrRpm1 were infiltrated into Arabidopsis Col-0 leaves, and cAMP amounts were determined. T3E injection was severely restricted when DC3000 expressed AvrRpm1, suggesting that ETI contributed to T3E injection restriction. The experiment was repeated three times with similar results, and se bars are shown.
Figure 7.
Figure 7.
Transgenic expression of PTI-suppressing T3Es in Arabidopsis relieves PTI-induced injection restriction. Arabidopsis wild-type plants and Arabidopsis transgenic plants expressing the DC3000 T3Es HopAO1-HA or HopF2-HA were pretreated with 1 μm flg21 for indicated time periods and infiltrated with 3 × 108 cells/mL of P. fluorescens(pLN1965) expressing HopU1-CyaA. The higher amounts of cAMP in PTI-induced transgenic plants compared to the wild-type control indicated that these T3Es can relieve PTI-induced injection restriction. This experiment was repeated at least three times with similar results, and se bars are shown.
Figure 8.
Figure 8.
PTI induced by hrcC inhibits the HR and restricts the injection longer than PTI-induced by flg21. A, PTI was induced in tobacco leaves by infiltration with 1 μm flg21 or 3 × 108 cells/mL of hrcC 0, 1, 2, 3, 5, 7, 10, and 14 d prior to an overlapping infiltration with 2 × 107 cells/mL of DC3000. Pictures were taken 48 h after DC3000 infiltration. The fraction to the left of each image indicates the number of times that the HR was inhibited over the total number of times the assay was performed. B, PTI was induced in tobacco leaves by infiltration with 1 μm flg21 or 3 × 108 cells/mL of hrcC 0, 1, 2, 3, 5, 7, 10, and 14 d prior to an overlapping infiltration with 3 × 108 cells/mL of DC3000(hopU1-cyaA). cAMP levels were measured 7 h after DC3000(hopU1-cyaA) infiltration. PTI induced by flg21 no longer inhibited the HR or restricted T3E injection after 2 d, whereas PTI induced by the hrcC mutant inhibited the HR and restricted injection throughout the 14-d experiment. These experiments were repeated twice with similar results, and se bars are shown.
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