2015 Jun 10
Pseudomonas Syringae Pv. Tomato DC3000 Type III Secretion Effector Polymutants Reveal an Interplay Between HopAD1 and AvrPtoB
Item in Clipboard
Pseudomonas Syringae Pv. Tomato DC3000 Type III Secretion Effector Polymutants Reveal an Interplay Between HopAD1 and AvrPtoB
Cell Host Microbe
The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered plant innate immune system by injecting a complex repertoire of type III secretion effector (T3E) proteins. Beyond redundancy and interplay, individual T3Es may interact with multiple immunity-associated proteins, rendering their analysis challenging. We constructed a Pst DC3000 polymutant lacking all 36 T3Es and restored individual T3Es or their mutants to explore the interplay among T3Es. The weakly expressed T3E HopAD1 was sufficient to elicit immunity-associated cell death in Nicotiana benthamiana. HopAD1-induced cell death was suppressed partially by native AvrPtoB and completely by AvrPtoBM3, which has mutations disrupting its E3 ubiquitin ligase domain and two known domains for interacting with immunity-associated kinases. AvrPtoBM3 also gained the ability to interact with the immunity-kinase MKK2, which is required for HopAD1-dependent cell death. Thus, AvrPtoB has alternative, competing mechanisms for suppressing effector-triggered plant immunity. This approach allows the deconvolution of individual T3E activities.
Copyright © 2015 Elsevier Inc. All rights reserved.
Figure 1. Deletion of 8 additional T3E genes from DC3000D28E produces effectorless mutant DC3000D36E and reveals that HopAD1 is responsible for the residual ability of DC300028E to elicit cell death and to suppress a ROS burst and PTI in
(A) Overview of the
Pst DC3000 T3E gene repertoire and deletions in polymutant strains. (B) ETI-like rapid, confluent, plant cell death is triggered in N. benthamiana by Pst DC3000 polymutants that still carry hopAD1. Bacteria suspensions at the high inoculum level of 5 ×10 8 CFU/ml were infiltrated into the marked zones of N. benthamiana leaves. The leaf response was photographed 2 days later. The fraction under each image indicates the number of times that tissue death was observed relative to the number of test inoculations. (C) ROS production is triggered in N. benthamiana by Pst DC3000 polymutants that lack hopAD1. Bacterial suspensions at 5 ×10 8 CFU/ml were infiltrated into leaves, and ROS production was determined 15 h post-inoculation. Means marked with the same letter were not significantly different at the 5% confidence level on the basis of Duncan’s multiple-range test. (D) PTI elicitation activity is restored to DC3000D28E derivatives by deletion of hopAD1. The fraction under each image indicates the number of times cell death was inhibited (indicating PTI elicitation by the first inoculum) relative to the number of test inoculations. All experiments were repeated three times with similar results (see also Table S1, Table S2 and Figure S1).
Figure 2. Evidence that HopAD1 is an avirulence determinant in
N. benthamiana based on heterologous expression of hopAD1 in P. syringae pv. tabaci ( Pta)11528 and deletion of hopAD1 from Pst DC3000
Pta 11528 expressing HopAD1 is avirulent in N. benthamiana. Leaves were infiltrated with the indicated strains at the low level of 3×10 4 CFU/ml and photographed 8 days later. (B) Pst DC3000 ΔhopAD1 mutants cause disease lesions in N. benthamiana. The leaves were infiltrated with the indicated strains at 3×10 4 CFU/ml using a blunt syringe and photographed 8 days after inoculation. (C) Deletion of hopAD1 from Pst DC3000 results in the same high level of growth as observed with Pst DC3000 ΔhopQ1-1 in N. benthamiana. Bacteria were infiltrated at 3×10 4 CFU/ml and populations were measured from three 0.6-cm-diameter leaf discs 6 days after inoculation. Error bars indicate the standard deviation of populations measured from thee leaf discs from each of three plants. Means marked with the same letter were not significantly different at the 5% confidence level on the basis of Duncan’s multiple-range test. All experiments were repeated three times with similar results (see also Figures S2 and S3).
Figure 3. Deletion of
hopAD1 has no effect on the growth or symptom production in N. benthamiana of DC3000D28E derivatives carrying T3E gene subsets that include avrPtoB, and AvrPtoB is sufficient to reduce cell death elicitation by HopAD1
(A) Bacterial growth assay by DC3000D28E derivatives with different subsets of effectors (Table S1). White fill in the genotype grid indicates that the locus was present or restored in DC3000D28E; gray fill indicates that it was missing. Bacteria were infiltrated at the low inoculum level of 3×10
4 CFU/ml, and populations in three 0.6-cm-diameter leaf discs were determined at 6 days after inoculation. Error bars indicate the standard deviation of populations measured from three leaf discs from each of three plants. Means with the same letter were not significantly different at the 5% confidence level based on Duncan’s multiple range test. (B) Symptom production by DC3000D28E derivatives with different subsets of effectors. The indicated bacteria were inoculated as above, and leaves were photographed at 8 days post inoculation. Asterisks indicate necrosis, plus signs indicate chlorosis, and minus signs indicate no symptoms. (C) AvrPtoB but not AvrPto suppresses cell death elicitation by HopAD1 in N. benthamiana. Leaves were infiltrated with indicated bacteria in the marked circles at the high inoculum level of 5×10 8 CFU/ml, and 2 days later they were photographed and scored for tissue collapse (unstained) or stained with trypan blue to assay cell death (stained). The fraction under each image indicates the number of times that confluent tissue collapse or trypan blue staining was observed relative to the number of test inoculations. All experiments were repeated three times with similar results (see also Figure S4).
Figure 4. AvrPtoB homologs from diverse
P. syringae pathovars, expressed in DC3000D28E, suppress HopAD1-dependent cell death elicitation in N. benthamiana, and their suppression efficacy correlates with having an inactive or missing E3 ligase domain
(A) Bacterial cell suspensions in MgCl
2 buffer, adjusted to 5×10 8 CFU/ml, were infiltrated into leaves, and the plant response was photographed 2 days later. The fraction under each image indicates the number of times tissue collapse occurred relative to the number of test inoculations. All avrPtoB homologs ( hopAB family) were expressed from the avrPto promoter in pCPP5372. The experiment was repeated three times with similar results (see also text and Figure S5). (B) Interaction of HopAB family members with Pto and Fen based on a yeast two-hybrid system. Blue patches indicate positive interactions.
Figure 5. AvrPtoB mutants show that suppression of cell death elicitation by HopAD1 is enhanced by mutation of the AvrPtoB E3 ligase domain and correlates with AvrPtoB interaction with MKK2 but not Pto or Fen, and expression of AvrPtoB
M3 in wild-type Pst DC3000 promotes bacterial growth and symptom production in N. benthamiana
(A) Diagram of AvrPtoB from
Pst DC3000 showing the type III secretion system targeting region (T3S) and mutations ablating function of the Pto interaction domain (PID), Fen interaction domain (FID), and E3 ubiquitin ligase domain (E3). (B) Effect of mutations in AvrPtoB domains on relative ability to suppress cell death elicitation in N. benthamiana by HopAD1 in DC3000D28E. Bacteria at the high inoculum level of 5×10 8 CFU/ml were infiltrated into leaves, and the plant response was photographed 4 days later. The fraction under each image indicates the frequency of tissue collapse relative to the number of test inoculations. Similar results were found in three independent tests. (C) Interaction of AvrPtoB mutants with Pto, Fen, and MKK2 based on a yeast two-hybrid system. Blue patches indicate positive interactions. (D) Bacteria were inoculated at the low level of 3×10 4 CFU/ml in N. benthamiana, and populations were assayed 6 days later. Error bars indicate the standard deviation of populations measured from thee leaf discs from each of three plants. Means with the same letter were not significantly different at the 5% confidence level based on Duncan’s multiple-range test. (E) N. benthamiana leaves were inoculated as above and photographed 15 days later. Typical symptoms are shown, and all the experiments were repeated three times with similar results.
Figure 6. Suppression of ETI by AvrPtoB
M3 action on MKK2 is supported by the ability of AvrPtoB M3 to inhibit AvrPto and AvrPtoB 1-387 cell death elicitation in Pto + N. benthamiana (despite AvrPtoB M3 lacking a functional Pto interaction domain) and by the MKK2-dependence of HopAD1 cell death elicitation
M3 suppresses cell death elicitation by avrPto + or AvrPtoB 1-387 + DC3000D29E in N. benthamiana that is transgenically producing the tomato Pto kinase. Leaves were infiltrated with indicated bacteria in the marked circles at the high inoculum level of 5×10 8 CFU/ml and stained with trypan blue at 2 days post inoculation. The fraction under each image indicates the number of times cell death was observed relative to the number of test inoculations. (B) Cell death elicited by HopAD1 in DC300028E is compromised by silencing MKK2 but not Prf in N. benthamiana, but cell death elicited by HopQ1-1 in DC3000D29E is not suppressed by MKK2 or Prf. Bacteria at 5×10 8 CFU/ml were infiltrated into leaves, and the plant response was photographed 2 days later. The fraction under each image indicates the number of times that tissue collapse was observed relative to the number of test inoculations. Similar results were found in three independent experiments.
Figure 7. Model of the competing virulence activities of AvrPtoB and AvrPtoB
M3 in the context of plant immune signaling, relevant activities of HopAD1 and AvrPto, and the domain structure of AvrPtoB
(A) HopAD1 is proposed to disrupt PTI-associated kinase signaling but be detectable by an unknown resistance protein (R1) that signals through MKK2 to ETI. AvrPtoB suppresses PTI through interaction of PID and FID domains with kinase domains of pattern recognition co-receptors (PRRs), FLS2, BAK1, and Bti9, but recognition by Fen/Pto decoy kinases leads to ETI unless blocked by E3 ligase action at the FID domain. The M3 mutant of AvrPtoB, lacking kinase-interaction and E3 ligase domains, has enhanced ability to interact with MKK2 and suppress ETI elicited by HopAD1, AvrPto, and AvrPtoB
1-387. High polymorphism in the E3 ligase domain of HopAB family members suggests that these effectors may rapidly evolve between states favoring E3 ligase activity (for suppression of FID-Fen-triggered ETI) or loss of such activity (for suppression of ETI triggered by HopAD1 and other effectors that signal through MKK2) depending on the host R gene composition. (B) Depiction of the AvrPtoB activities described above in terms of AvrPtoB domains and interacting host proteins highlights the conflicting roles of the M3 activity (within gray), the PID/FID domains, and the E3 ubiquitin ligase activity. Outcomes involving different AvrPtoB variants and R proteins have been previously reported (black font)(Mathieu et al., 2014; Rosebrock et al., 2007) or shown here (blue font). Variants a-c depict the conflicting activity of the E3 ubiquitin ligase domain in suppression of intramolecular (self domain) ETI versus ETI elicited by one or more other effectors signaling through MKK2. Variants c-e address the ability of AvrPtoB M3 (e) to suppress Pto-dependent ETI elicitation by AvrPtoB 1-387 (d), which demonstrates that PID/FID-dependent interaction with Pto (but not with PRRs) conflicts with the ability of AvrPtoB variants to suppress MKK2-dependent ETI elicitation (See also Figures S6 and S7).
All figures (7)
Modular Study of the Type III Effector Repertoire in Pseudomonas Syringae Pv. Tomato DC3000 Reveals a Matrix of Effector Interplay in Pathogenesis
HL Wei et al.
Cell Rep 23 (6), 1630-1638.
The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered innate immune system of Nicotiana benthamiana and other plants by injecting a comp …
Plant Immunity Directly or Indirectly Restricts the Injection of Type III Effectors by the Pseudomonas Syringae Type III Secretion System
E Crabill et al.
Plant Physiol 154 (1), 233-44.
Plants perceive microorganisms by recognizing microbial molecules known as pathogen-associated molecular patterns (PAMPs) inducing PAMP-triggered immunity (PTI) or by rec …
A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity
B Zhou et al.
Plant Physiol 173 (2), 1371-1390.
Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in …
Defining Essential Processes in Plant Pathogenesis With Pseudomonas Syringae Pv. Tomato DC3000 Disarmed Polymutants and a Subset of Key Type III Effectors
HL Wei et al.
Mol Plant Pathol 19 (7), 1779-1794.
Pseudomonas syringae pv. tomato DC3000 and its derivatives cause disease in tomato, Arabidopsis and Nicotiana benthamiana. The primary virulence factors include a reperto …
AvrPtoB: A Bacterial Type III Effector That Both Elicits and Suppresses Programmed Cell Death Associated With Plant Immunity
RB Abramovitch et al.
FEMS Microbiol Lett 245 (1), 1-8.
Pseudomonas syringae pv. tomato DC3000 is a model pathogen for studying the molecular basis of plant immunity and disease susceptibility in tomato and Arabidopsis. DC3000 …
PubMed Central articles
Pseudomonas Syringae AlgU Downregulates Flagellin Gene Expression, Helping Evade Plant Immunity
Z Bao et al.
J Bacteriol 202 (4).
Flagella power bacterial movement through liquids and over surfaces to access or avoid certain environmental conditions, ultimately increasing a cell's probability of sur …
Pangenomic Type III Effector Database of the Plant Pathogenic
CRR Sabbagh et al.
PeerJ 7, e7346.
We curated the T3E repertoires of 12 plant pathogenic
Ralstonia strains, representing a total of 12 strains spread over the different groups of the species complex …
Pseudomonas Syringae Effector AvrPtoB Associates With and Ubiquitinates Arabidopsis Exocyst Subunit EXO70B1
W Wang et al.
Front Plant Sci 10, 1027.
Many bacterial pathogens secret effectors into host cells to disable host defenses and thus promote infection. The exocyst complex functions in the transport and secretio …
Identification of Benzyloxy Carbonimidoyl Dicyanide Derivatives as Novel Type III Secretion System Inhibitors
via High-Throughput Screening
YN Ma et al.
Front Plant Sci 10, 1059.
The type III secretion system (T3SS) in many Gram-negative bacterial pathogens is regarded as the most critical virulence determinant and an attractive target for novel a …
Post-Translational Modifications of Proteins Have Versatile Roles in Regulating Plant Immune Responses
J Yin et al.
Int J Mol Sci 20 (11).
To protect themselves from pathogens, plants have developed an effective innate immune system. Plants recognize pathogens and then rapidly alter signaling pathways within …
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Gene Expression Regulation, Bacterial
MAP Kinase Kinase 2 / genetics
MAP Kinase Kinase 2 / metabolism
Plant Proteins / genetics
Plant Proteins / metabolism
Plants, Genetically Modified
Protein Structure, Tertiary
Protein-Serine-Threonine Kinases / genetics
Protein-Serine-Threonine Kinases / metabolism
Pseudomonas syringae / genetics
Pseudomonas syringae / pathogenicity
Pseudomonas syringae / physiology
Reactive Oxygen Species / metabolism
Ubiquitin-Protein Ligases / metabolism
avrPto protein, Pseudomonas syringae
Pto protein, Lycopersicon
LinkOut - more resources
Full Text Sources Research Materials Miscellaneous