Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 8, 2157
eCollection

Pseudomonas syringae Pv. actinidiae Type III Effectors Localized at Multiple Cellular Compartments Activate or Suppress Innate Immune Responses in Nicotiana benthamiana

Affiliations

Pseudomonas syringae Pv. actinidiae Type III Effectors Localized at Multiple Cellular Compartments Activate or Suppress Innate Immune Responses in Nicotiana benthamiana

Sera Choi et al. Front Plant Sci.

Abstract

Bacterial phytopathogen type III secreted (T3S) effectors have been strongly implicated in altering the interaction of pathogens with host plants. Therefore, it is useful to characterize the whole effector repertoire of a pathogen to understand the interplay of effectors in plants. Pseudomonas syringae pv. actinidiae is a causal agent of kiwifruit canker disease. In this study, we generated an Agrobacterium-mediated transient expression library of YFP-tagged T3S effectors from two strains of Psa, Psa-NZ V13 and Psa-NZ LV5, in order to gain insight into their mode of action in Nicotiana tabacum and N. benthamiana. Determining the subcellular localization of effectors gives an indication of the possible host targets of effectors. A confocal microscopy assay detecting YFP-tagged Psa effectors revealed that the nucleus, cytoplasm and cell periphery are major targets of Psa effectors. Agrobacterium-mediated transient expression of multiple Psa effectors induced HR-like cell death (HCD) in Nicotiana spp., suggesting that multiple Psa effectors may be recognized by Nicotiana spp.. Virus-induced gene silencing (VIGS) of several known plant immune regulators, EDS1, NDR1, or SGT1 specified the requirement of SGT1 in HCD induced by several Psa effectors in N. benthamiana. In addition, the suppression activity of Psa effectors on HCD-inducing proteins and PTI was assessed. Psa effectors showed differential suppression activities on each HCD inducer or PTI. Taken together, our Psa effector repertoire analysis highlights the great diversity of T3S effector functions in planta.

Keywords: Nicotiana benthamiana; Nicotiana tabacum; avirulence; effector screening; hypersensitive response; subcellular localization; virus-induced gene silencing.

Figures

FIGURE 1
FIGURE 1
Subcellular localization of Psa effectors in Nicotiana benthamiana. Four–five week-old N. benthamiana leaf cells were infected with Agrobacterium AGL1 carrying C-terminally YFP-tagged Psa effectors for transient protein expression. At 2 days post infection (dpi), 8 mm diameter leaf disks were sampled and viewed using confocal microscopy. YFP fluorescence was excited at 488 nm with a 20 mW Argon laser and captured in the emission range between 500 and 530 nm. HopM1 YFP signal was determined by a sum-of-squares Z-projection. Chloroplast auto-fluorescence was detected between 600 and 680 nm. This experiment was repeated twice with similar results.
FIGURE 2
FIGURE 2
Multiple Psa effectors trigger HR-like cell death in Nicotiana spp. Four–five weeks-old N. benthamiana and N. tabacum plants were infiltrated with Agrobacterium tumefaciens AGL1 strains carrying C-terminally YFP-tagged Psa effector constructs. Photographs of the leaves showing HR-like cell death (HCD) were taken at 3 dpi. Leaf images showing HCD are indicated with a red border. (+), (++), or (+++) indicate the strength of the HCD phenotype as measured by ion conductivity (Supplementary Figures S3, S4). This experiment was conducted at least three times with similar results.
FIGURE 3
FIGURE 3
NbSGT1 is partially required for HR triggered by several Psa effectors. (A) RPS4TIR-triggered HCD in EV–, NbEDS1–, NbNDR1–, or NbSGT1–silenced N. benthamiana plants. Two-week old N. benthamiana plants were infiltrated with mixture of A. tumefaciens AGL1 strains carrying pTRV1 or pTRV2 construct as indicated in section “Materials and Methods.” Four weeks later, plants were agroinfiltrated to express the RPS4 TIR domain (RPS4TIR). RPS4TIR-induced HCD was scored at 4 dpi. The number of HCD-showing patches out of total infiltrated patches are indicated under each panel. Numbers labeled in red indicate a significant reduction of HCD. (B) Psa effector-triggered HCD in NbEDS1–, NbNDR1–, and NbSGT1–silenced N. benthamiana plants. VIGS and agroinfiltration to induce HCD are indicated in (A). Psa effector-induced HCD was scored at 4 dpi. The number of HCD-showing patches out of total infiltrated patches are indicated under each panel. Numbers labeled in red indicate a significant reduction of HCD. (C) Semi-quantitative PCR amplification of silenced genes in VIGS plants. 8 mm diameter leaf disks were harvested from VIGSed plants, total RNA was extracted by Trizol RNA method and cDNA was synthesized. Using cDNA as PCR template, partial fragments of Actin (control) and VIGSed genes were amplified with gene-specific primers.
FIGURE 4
FIGURE 4
Multiple Psa effectors suppress HCD triggered by different effectors in N. benthamiana. (A) Psa effectors suppress BAX-induced HCD in N. benthamiana. Agrobacterium strains were mixed in a 1:4 ratio (BAX:Psa effector or GFP). (B) Psa effectors suppress AvrPto/Pto-induced HCD in N. benthamiana. Agrobacterium strains were mixed in 1:1:1 ratio (AvrPto:Pto:Psa effector or GFP). (C) Suppression of Psa effector-induced HCD in N. benthamiana. Agrobacterium strains were mixed in a 1:4 ratio (HCD-inducing effector:HCD-suppressing effector). Four–five week-old N. benthamiana leaves were agroinfiltrated and HCD was photographed at 3 (A,B) or 5 (C) dpi. This experiment was repeated more than three times with similar results.
FIGURE 5
FIGURE 5
The Psa effector HopD1 interferes with Pf Pf0-1(T3S)-induced suppression of Pto DC3000-triggered HCD. Leaves from 4 to 5 week-old N. benthamiana plants were infiltrated with Pf Pf0-1(T3S) carrying popP2, popP2-C321A or hopD1 (2 × 107 CFU/mL) 8 h prior to Pto DC3000 (3 × 108 CFU/mL) infection. Pto DC3000-triggered cell death was scored and photographed at 48 h post Pto DC3000 infection.

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

References

    1. Aarts N., Metz M., Holub E., Staskawicz B. J., Daniels M. J., Parker J. E. (1998). Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signaling pathways in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 95 10306–10311. 10.1073/pnas.95.17.10306 - DOI - PMC - PubMed
    1. Abramovitch R. B. (2003). Pseudomonas type III effector AvrPtoB induces plant disease susceptibility by inhibition of host programmed cell death. EMBO J. 22 60–69. 10.1093/emboj/cdg006 - DOI - PMC - PubMed
    1. Abramovitch R. B., Janjusevic R., Stebbins C. E., Martin G. B. (2006). Type III effector AvrPtoB requires intrinsic E3 ubiquitin ligase activity to suppress plant cell death and immunity. Proc. Natl. Acad. Sci. U.S.A. 103 2851–2856. 10.1073/pnas.0507892103 - DOI - PMC - PubMed
    1. Adlung N., Bonas U. (2017). Dissecting virulence function from recognition: cell death suppression in Nicotiana benthamiana by XopQ/HopQ1-family effectors relies on EDS1-dependent immunity. Plant J. 91 430–442. 10.1111/tpj.13578 - DOI - PubMed
    1. Adlung N., Prochaska H., Thieme S., Banik A., Blüher D., John P., et al. (2016). Non-host resistance induced by the Xanthomonas effector XopQ is widespread within the genus Nicotiana and functionally depends on EDS1. Front. Plant Sci. 7:1796. 10.3389/fpls.2016.01796 - DOI - PMC - PubMed
Feedback