You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Introduction {#s1}
============

Plants have evolved a multilayered immune system to cope with pathogens. For the initial immune response, conserved pathogen-derived molecules, termed pathogen-associated molecular patterns (PAMPs), are detected at the plant cell surface by the plasma membrane (PM)-bound receptor-like kinases (RLKs) or receptor-like proteins (RLPs), termed pattern recognition receptors (PRRs) ([@B57]; [@B52]). Well-studied PRRs include the *Arabidopsis* leucine-rich repeat receptor kinases FLAGELLIN SENSING 2 (FLS2) and EF-TU RECEPTOR (EFR), which recognize bacterial flagellin and EF-Tu, respectively ([@B70]; [@B69]). After perception of PAMPs by the PRRs, a series of defense responses are activated, such as a burst of reactive oxygen species (ROS), an increase of Ca^2+^ concentration, callose deposition, mitogen-activated protein kinase (MAPK) activation, and pathogenesis-related gene expression, termed PAMP-triggered immunity (PTI) ([@B26]; [@B52]). The BRASSINOSTEROID RECEPTOR-ASSOCIATED KINASE 1 (BAK1) interacts with FLS2 or EFR and functions as a co-receptor in PTI ([@B8]; [@B50]).

To aid in infection, pathogens suppress PTI by secreting virulence effectors into the intercellular space or the cytoplasm of host cells and cause effector-triggered susceptibility (ETS) ([@B26]). In the second layer of innate immunity, termed effector-triggered immunity (ETI), pathogen effectors are directly or indirectly recognized by the host mainly *via* intracellular nucleotide-binding (NB) leucine-rich repeat (LRR) domain receptors (NLRs); recognition of effectors by NLR proteins leads to robust immunity that terminates pathogen growth ([@B10]; [@B15]).

The gram-negative plant bacterial pathogen *Pseudomonas syringae* pv. *tomato* (*Pto*) strain DC3000 causes bacterial speck disease in tomato (*Solanum lycopersicum*) and *Arabidopsis thaliana* and injects about 30 effectors *via* the type III secretion system into host cells to suppress plant innate immunity ([@B39]; [@B53]). AvrPto and AvrPtoB are two well-analyzed, sequence-distinct effectors from *Pto* DC3000. In tomato, both AvrPto and AvrPtoB interact with the tomato protein Pto, a serine/threonine protein kinase, and elicit Prf-mediated programmed cell death (PCD) and immunity ([@B29]; [@B60]). AvrPtoB also targets a host protein kinase, Fen, to disrupt plant immunity ([@B41]). In *Arabidopsis*, AvrPto and AvrPtoB suppress basal defense and intercept multiple PAMPs-mediated signaling upstream of MAPKKK ([@B12]; [@B24]). AvrPto and AvrPtoB also can induce endogenous proteolytic activity that degrades RESISTANCE TO PSEUDOMONAS SYRINGAE PV MACULICOLA 1 (RPM1)-INTERACTING PROTEIN 4 (RIN4) in the presence of Pto and Prf ([@B34]). In *Arabidopsis* and tomato, AvrPto suppresses cell wall-based defenses and AvrPtoB promotes the virulence of *Pto* DC3000 by suppressing PCD ([@B2]; [@B1]; [@B59]). AvrPto and AvrPtoB target multiple RLKs involved in the perception of PAMPs in *Arabidopsis*. For instance, AvrPto and AvrPtoB target BAK1 to interfere with the bacterial-induced formation of the FLS2-BAK1 receptor--signaling complex ([@B46]; [@B7]). AvrPto targets BOTRYTIS-INDUCED KINASE 1 (BIK1), a downstream component of the FLS2 pathway, to prevent the phosphorylation of BIK1 ([@B58]). AvrPtoB contains an N-terminal region at 1--307 amino acids (aa), which is sufficient to elicit Pto/Prf-mediated PCD ([@B60]), and a C-terminal U-box type E3 ubiquitin ligase domain. AvrPtoB targets and degrades FLS2, EFR, and the LysM domain receptor kinase CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) to block the initiation of PTI signaling in *Arabidopsis* ([@B19]; [@B18]). AvrPtoB ubiquitinates and degrades NON-EXPRESSER OF PR GENES 1 (NPR1) to block salicylic acid-dependent transcriptional reprogramming ([@B6]).

Targeted protein transport and coordinated membrane dynamics play an important role in PTI ([@B20]). These trafficking events are mediated by a large group of regulatory proteins, such as the exocyst complex, which may function in the transport of defense molecules and contributes to their secretion ([@B40]; [@B37]). EXO70 is one of the eight subunits of the exocyst complex. The *Arabidopsis* genome encodes 23 EXO70 protein family members, some of which are involved in plant immunity ([@B11]). For instance, the *exo70B1* loss-of-function mutant displays enhanced resistance to the powdery mildew pathogen *Golovinomyces cichoracearum*, the bacterial pathogen *Pto* DC3000, and the oomycete pathogen *Hyaloperonospora arabidopsidis* Noco2 ([@B67]). EXO70B1 is also implicated in autophagy-related transport to the vacuole, and loss of function of EXO70B1 causes reduced numbers of internalized autophagosomes inside the vacuole ([@B30]), along with ectopic hypersensitive responses ([@B30]; [@B49]; [@B67]). In addition, EXO70B1 positively regulates light- and drought-induced stomatal movement ([@B25]; [@B45]), and EXO70B1 is co-localized with EXO84B in broad bean (*Vicia faba*) guard cells ([@B25]). EXO70B2 and EXO70B1 show the highest sequence similarity, and both have been reported to be necessary for PTI and resistance to pathogens ([@B48]; [@B49]). Moreover, RIN4 recruits EXO70B1 to the plant cell PM ([@B44]). The plant U-box-type ubiquitin ligase 22 (PUB22) ubiquitinates and degrades EXO70B2, which contributes to PAMP-triggered responses ([@B48]).

The activated immune responses in the *exo70B1* mutant require the atypical NLR protein Toll/interleukin-1 receptor--nucleotide-binding sequence protein (TIR-NBS2; herein referred to as TN2) and CALCIUM-DEPENDENT PROTEIN KINASE 5 (CPK5). Overexpression of CPK5 also leads to TN2-dependent autoimmune responses. Moreover, TN2 directly interacts with EXO70B1, and the expression level of *TN2* is up-regulated in mature *exo70B1-3* mutants ([@B67]; [@B32]). TN2 belongs to the TIR-NBS (TN) family, which has 21 members in *Arabidopsis* ecotype Col-0 ([@B36]; [@B27]). However, how TN2 contributes to *exo70B1*-activated resistance is still unclear. We proposed that EXO70B1 is a guardee of TN2 or the TN2-related complex and NLR-mediated immunity is activated when EXO70B1 is degraded ([@B67]; [@B32]). To date, only two effectors are reported to target the components of the exocyst complex. In potato (*Solanum tuberosum*), *Phytophthora infestans* manipulates plant immunity by targeting the exocyst component Sec5 with the RXLR effector AVR1 ([@B14]). In rice (*Oryza sativa*), the effector Avr-Pii from the rice blast fungus *Magnaporthe oryzae* interacts with OsEXO70F2 and OsEXO70F3, and the rice NLR protein Pii recognizes Avr-Pii and triggers OsEXO70F3-dependent immunity ([@B17]).

Here, we show that EXO70B1 contributes to plant immunity triggered by multiple PAMPs and AvrPtoB interacts with EXO70B1 *via* its N-terminal domain. AvrPtoB ubiquitinates and mediates the degradation of EXO70B1. Overexpression of AvrPtoB in *Arabidopsis* leads to autoimmune phenotypes, which are partially dependent on TN2. AvrPtoB contributes to the virulence of *Pto* DC3000 by overcoming EXO70B1-mediated resistance. AvrPtoB can rescue TN2- or TN2-TIR-triggered cell death in tobacco, which is suppressed by EXO70B1. Taken together, these findings indicate that EXO70B1 functions positively in PTI signaling and is a substrate of AvrPtoB.

Materials and Methods {#s2}
=====================

Plant Materials and Growth Conditions {#s2_1}
-------------------------------------

The *Arabidopsis thaliana tn2-10* (SALK_204239C) mutant was obtained from the *Arabidopsis* Biological Resource Center. The *exo70B1-3* mutant and the *exo70B1-3* transgenic line containing a construct expressing EXO70B1-GFP under the control of the native *EXO70B1* promoter were described previously ([@B67]). Double mutants were obtained by genetic crosses and identified by polymerase chain reaction (PCR). *Arabidopsis* plants were grown in a growth room at 20°C to 22°C and ∼60% relative humidity with a 16/8-h day/night photoperiod for seed setting and a 9/15-h day/night photoperiod for phenotyping, with a light intensity of 7,000 to 8,000 lux. *Nicotiana tabacum* and *Nicotiana benthamiana* plants were grown under the same short-day conditions as *Arabidopsis* ([@B56]).

Vector Construction and Plant Transformation {#s2_2}
--------------------------------------------

The *AvrPtoB* sequence was amplified by PCR from *Pto* DC3000 genomic DNA and inserted into the pEASY vector using a pEASY Simple blunt Clonase Kit (TransGen Biotech). The *AvrPtoB^F479A^* mutant form was created using site-directed mutagenesis as described previously ([@B68]). The *AvrPtoB* sequence was amplified with *Kpn*I and *Sal*I restriction sites and cloned into pSuper1300 with a 6 × Myc tag at the C-terminal to generate the*35S:AvrPtoB-Myc* construct ([@B66]). The *Agrobacterium tumefaciens* strain GV3101 carrying the construct was used to transform *Arabidopsis* plants by floral-dip transformation ([@B9]). The stably transformed plants carrying a single copy of the insert were identified in the T~3~ generation, and two independent stable transgenic lines AvrPtoB-Myc \#2 and AvrPtoB-Myc \#7 were chosen for further analysis.

ROS Assays {#s2_3}
----------

ROS assays were performed as described previously ([@B64]). Leaf strips of 4-week-old plants were treated with 100 nM of flg22, 100 nM of elf18, or 0.1 mg/ml of chitin. Luminescence was detected with the GloMax 96 microplate luminometer (Promega).

Callose Deposition {#s2_4}
------------------

Leaves from 4-week-old *Arabidopsis* plants were infiltrated with H~2~O, 20 μM of flg22, 20 μM of elf18, or 2 mg/ml of chitin in 10 mM of MgCl~2~ and removed 12 h after infiltration. Callose staining, image acquisition, and processing were carried out as described ([@B65]).

Y2H Assay {#s2_5}
---------

The Matchmaker GAL4 Two-Hybrid System 3 (Clontech) was used for yeast two-hybrid (Y2H) assays. Bacterial effector sequences were amplified from *Pto* DC3000 genomic DNA and were cloned into the vector pGBKT-7. The full-length *EXO70B1*, *EXO70B2*, and *EXO70A1* cDNA was cloned into pGADT-7. Different pairs of constructs were cotransformed into AH109, and the cells were grown on SD-Trp/-Leu medium. Three clones from SD-Trp/-Leu plates were transferred to SD-Trp/-Leu/-His/-Ade plates. Single clones were incubated in SD-Trp/-Leu liquid medium, and 10 μl of suspensions (diluted to OD~600~ = 0.5) of the overnight cultures was dropped onto different plates to test the interactions of the proteins.

RT-qPCR {#s2_6}
-------

Total RNA extraction and quantitative reverse transcription PCR (RT-qPCR) were performed as described previously ([@B47]).

Pull-Down Assay {#s2_7}
---------------

The pull-down assay was performed as described previously ([@B62]). The *AvrPtoB* and *AvrPtoB~1--410~* sequences were amplified from *AvrPtoB-pEASY*, and the *AvrPtoB^F479A^* sequence was amplified from *AvrPtoB^F479A^-pEASY* with *Eco*RI and *Not*I restriction sites and were cloned into the prokaryotic expression vector pGEX-4T-1. The positive clones were selected by sequencing. The GST-AvrPtoB, GST-AvrPtoB^F479A^, and GST-AvrPtoB~1--410~ fusion proteins were expressed in *Escherichia coli* as described previously ([@B61]). The *EXO70B1* sequence was amplified from the *EXO70B1-AD* plasmid with *Kpn*I and *Sal*I restriction sites and cloned into pSuper1300 with a GFP tag at the C-terminal. EXO70B1-GFP was transiently expressed in *N. benthamiana* leaves by *Agrobacterium tumefaciens*-mediated infiltration at OD~600~ = 1.5, and samples were collected after 2 days. EXO70B1-GFP was extracted in native extraction buffer as described previously ([@B47]). Equimolar amounts of glutathione *S*-transferase (GST), GST-AvrPtoB, GST-AvrPtoB^F479A^, and GST-AvrPtoB~1--410~ were incubated with equal amounts of EXO70B1-GFP extracts at 4°C for 4 h. After incubation, the GST binding beads (Novagen) were added to incubate for another 2 h. Then, the mix was washed three times using GST washing buffer (40 mM of Na~2~HPO~4~, 8.5 mM of KH~2~PO~4~, 1.4 M of NaCl, 27 mM of KCl \[pH 7.3\], and 5 mM of dithiothreitol \[DTT\]). After the last centrifugation, the GST washing buffer was removed. Eighty microliters of GST washing buffer and 20 μl of 5 × SDS sample buffer were added, and the beads were boiled for 10 min. The proteins were separated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) and then detected using anti-GST (Abmart) and anti-GFP (Abmart) antibodies.

Co-IP Assay in *N. Benthamiana* {#s2_8}
-------------------------------

Coimmunoprecipitation (Co-IP) assays were performed as described previously, with minor modifications ([@B47]). Two days after infiltration, leaves were syringe infiltrated with or without 50 μM of MG132 for 3 h before freezing in liquid nitrogen. For anti-Myc immunoprecipitation, protein extracts were incubated with agarose-conjugated anti-Myc antibody (MBL) for 4 h at 4°C with gentle rotation. The agarose beads were washed and resuspended in 80 μl of PBS and 20 μl of 5 × SDS sample buffer and boiled for 10 min. Protein samples were detected with anti-GFP (Abmart) and anti-Myc (Abmart) immunoblot.

Cell-Free and *In Vivo* Protein Degradation Assay {#s2_9}
-------------------------------------------------

The *35S:AvrPtoB-Myc* construct or an empty vector control construct was transiently expressed in *N. benthamiana* leaves by *Agrobacterium tumefaciens*-mediated infiltration at OD~600~ = 1.0, and samples were collected after 2 days. Total leaf proteins were extracted in native extraction buffer. The *EXO70B1* sequence was amplified from the *EXO70B1-AD* plasmid with *Bam*HI and *Sal*I restriction sites and were cloned into the prokaryotic expression vector pMAL-c2g-1, and the MBP-EXO70B1 fusion protein was expressed in *E. coli*. Maltose-binding protein (MBP) and the MBP-EXO70B1 recombinant protein were incubated with a cell-free crude extract for 1.5 and 3 h in the presence or absence of 50 μM of MG132 (AG Scientific). The reactions were terminated by adding 5 × SDS sample buffer. The protein degradation patterns were analyzed by immunoblotting with anti-MBP (New England Biolabs) antibody, and AvrPtoB-Myc protein was detected by anti-Myc (Abmart) antibody.

For the *in vivo* protein degradation assay, the *EXO70B1* coding sequence was inserted into the PUC19 vector with a HA tag using *Sac*I and *Sal*I restriction sites. The *35S:EXO70B1-HA* and *35S:GFP* plasmids were cotransfected with the *35S:AvrPtoB-Myc* or *35S:AvrPtoB^F479A^-Myc* construct into protoplasts prepared from rosette leaves of wild-type *Arabidopsis* plants using the polyethylene glycol (PEG) method ([@B43]). After incubation for 12 h, the protoplasts were treated with MG132 (50 μM) or dimethyl sulfoxide (DMSO) for 3 h. The total proteins were extracted in native extraction buffer, and then 5 × SDS sample buffer was added. The patterns of protein degradation were analyzed by immunoblot analysis with anti-HA (Abmart) and anti-GFP (Abmart) antibodies. AvrPtoB-Myc was detected by anti-Myc (Abmart) antibody.

*In Vitro* Ubiquitination {#s2_10}
-------------------------

*In vitro* ubiquitination was carried out as described ([@B18]; [@B48]). In brief, each reaction of 60 μl of final volume contained ubiquitination buffer (50 mM of Tris--HCl \[pH 7.5\], 5 mM of MgCl~2~, 2 mM of DTT, 4 mM of ATP, and 1 × protease inhibitor cocktail) and 1 μg of MBP-EXO70B1 or MBP-EXO70B2 in the presence or absence of 15 μg of ubiquitin, 50 ng of human E1 (UBE1), 200 ng of *Arabidopsis* E2 His-UBC8, and 500 ng of GST-AvrPtoB or GST-AvrPtoB^F479A^. The reactions were incubated at 30°C for 3 h and stopped by adding 5 × SDS sample buffer and boiling for 10 min. Samples were separated by an 8% SDS-PAGE gel followed by detection of ubiquitinated substrate by immunoblotting using anti-MBP (New England Biolabs) and anti-GST (Abmart) antibodies.

Pathogen Infection Assays {#s2_11}
-------------------------

Bacterial strains were grown overnight at 28°C in King's B medium with appropriate antibiotics. Seedling infection assays were performed as described previously ([@B33]). In brief, seedlings were grown in 1/2 MS liquid medium for 10 days in a 12-well tissue culture plate. Bacteria were added at the concentration of OD~600~ = 0.2. Bacterial counting was performed 3 days after inoculation.

For powdery mildew infection, *Golovinomyces cichoracearum* strain UCSC1 was used to infect *Arabidopsis* plants at a high spore density ([@B3]). The infected leaves were stained with Trypan Blue at 8 days postinfection to visualize hyphae and dead cells ([@B16]).

Gene Expression {#s2_12}
---------------

For gene expression analysis, *Arabidopsis* leaves of Col-0 plants were hand-infiltrated with *Pto* DC3000 or the *Pto ΔAvrPtoB* mutant at OD~600~ = 0.2 in 10 mM of MgCl~2~. Total RNA extraction and RT-qPCR were performed as described above.

Hypersensitive Response Assay {#s2_13}
-----------------------------

*Agrobacterium* strain GV3101 was injected into 4-week-old *N. benthamiana* or *N. tabacum* leaves. The injected leaves were removed at 4 days postinoculation and photographed, then stained using Trypan Blue to examine cell death, and photographed.

Oligonucleotide Sequences {#s2_14}
-------------------------

The primers used in this study are listed in [**Supplementary Table 1**](#SM1){ref-type="supplementary-material"}.

Results {#s3}
=======

EXO70B1 Associates With AvrPtoB {#s3_1}
-------------------------------

EXO70B1 is required for bacterial flagellin peptide 22 (flg22)-triggered immunity in plants ([@B48]). To further investigate the role of EXO70B1 in PTI, we examined elf18- and chitin-induced ROS production and callose deposition in the *exo70B1-3* mutant. When treated with elf18 or chitin, the*exo70B1-3* mutant showed reduced production of ROS than did the wild type Col-0 ([**Figures 1A--C**](#f1){ref-type="fig"}). Similarly, flg22-, elf18-, and chitin-induced callose deposition was also significantly reduced in the *exo70B1-3* mutant ([**Figure 1D**](#f1){ref-type="fig"}), indicating that EXO70B1 functions positively in multiple PAMPs-triggered responses in *Arabidopsis*.

![EXO70B1 is required for responses triggered by multiple PAMPs. **(A)** and **(B)** Luminescence-based assays of ROS production in leaves of four-week-old Col-0 and *exo70B1-3 Arabidopsis* plants following treatment with 100 nM of elf18 **(A)** or 0.1 mg/ml of chitin **(B)**. The means and standard deviations were determined using 12 independent biological samples. **(C)** Flg22-, elf18-, chitin-, and control (H~2~O)-induced ROS production over 30 min, presented as total photon counts in Col-0 and *exo70B1-3* mutants. Data represent the mean and standard deviation of eight independent biological samples. Asterisk indicates a statistically significant difference between Col-0 and *exo70B1-3* mutants (*P* \< 0.05; Student's *t*-test). **(D)** Flg22-, elf18-, chitin-, and control (H~2~O)-induced callose deposition in Col-0 and *exo70B1-3* mutants. Data represent the mean and standard deviation of 27 independent biological samples. Asterisk indicates a statistically significant difference between Col-0 and *exo70B1-3* mutants (*P* \< 0.05; Student's *t*-test). These experiments were repeated three times with similar results.](fpls-10-01027-g001){#f1}

Because of the positive role of EXO70B1 in PTI, we hypothesized that EXO70B1 may be targeted by pathogen effectors. To validate this hypothesis, we performed a Y2H screen to identify bacterial effectors that interact with EXO70B1. We identified several effectors from *Pto* DC3000 that interact with EXO70B1, including HopH1 ([@B54]), HopK1 ([@B23]), HopO1-1 ([@B21]), HopY1 ([@B22]), AvrPto ([@B29]; [@B24]), and AvrPtoB^F479A^, the E3 ligase-deficient mutant of AvrPtoB ([@B19]; [@B18]) ([**Table 1**](#T1){ref-type="table"}). According to our hypothesis, TN2 recognizes effectors by monitoring the status of EXO70B1, and the TN2-mediated resistance is activated when EXO70B1 disappeared ([@B67]; [@B32]). Then, we focused on AvrPtoB as AvrPtoB is a well-characterized effector, which contains an E3 ligase domain in its C-terminal. EXO70B1 interacted with the E3 ligase-deficient mutant form of AvrPtoB only, indicating that EXO70B1 may be a substrate of wild-type AvrPtoB.

###### 

EXO70B1 interacting effectors from *Pto* DC3000 identified by a Y2H screening.

  Effectors        Accession number   Size (aa)   Characterization             Virulence
  ---------------- ------------------ ----------- ---------------------------- -----------
  AvrPto           AAO57459           164         Unknown                      Yes
  AvrPtoB^F479A^   AY074795           553         E3 ligase deficient mutant   No
  HopH1            AAO54130           218         Protease                     Yes
  HopK1            AAO53599           338         Unknown                      No
  HopO1-1          AF458392           283         ADP-Ribosyltransferase       Yes
  HopY1            AF458403           287         Unknown                      Unknown

AvrPtoB contains two major domains and several specific domains in the N-terminus ([**Supplementary Figure S1A**](#SM1){ref-type="supplementary-material"}). AvrPtoB interacts with several target proteins through its N-terminal domains and degrades them *via* E3 ubiquitin ligase activity ([@B19]; [@B18]; [@B6]). To test whether AvrPtoB interacts with EXO70B1 *via* its N-terminal domains, we introduced constructs encoding the N-terminal region lacking the E3 ligase domain of AvrPtoB (AvrPtoB~1--410~) as well as the E3-ligase-deficient mutant AvrPtoB^F479A^ into yeast (*Saccharomyces cerevisiae*) along with constructs encoding EXO70B1. Both forms of AvrPtoB interacted with EXO70B1 ([**Figure 2A**](#f2){ref-type="fig"}), indicating that AvrPtoB maybe associated with EXO70B1 *via* one of the protein interacting domains in its N-terminal region. We did not observe that the full-length AvrPtoB interacts with EXO70B1 in Y2H assay, maybe because of the AvrPtoB E3 ligase activity, which may affect protein interaction in yeast.

![EXO70B1 associates with AvrPtoB. **(A)** EXO70B1 interacts with AvrPtoB in a Y2H assay. The coding sequences of AvrPtoB, AvrPtoB^F479A^, and AvrPtoB~1--410~ were fused to the Gal4 DNA binding domain (BD), and the coding sequence of EXO70B1 was fused to the Gal4 transactivation domain (AD). As a positive control, tomato Pto was also fused to the AD. Different pairs of constructs were cotransformed into AH109. A 10-μl suspension (OD~600~ = 0.5) of each cotransformant was dropped onto synthetic dropout (SD) medium lacking Leu and Trp and SD medium lacking Ade, His, Leu, and Trp. Photographs were taken after 5 days of incubation. **(B)** Amino acids 1--307 of AvrPtoB are necessary for the interaction with EXO70B1 in a Y2H assay. Different truncated fragments of AvrPtoB (1--205, 1--307, 1--387, and 308--553) were fused to the BD, and EXO70B1 and TN2 were fused to the AD. Different pairs of constructs were cotransformed into AH109. Yeast cells containing the indicated plasmids were spotted onto SD-Leu-Trp and SD-Ade-His-Leu-Trp. Photographs were taken after 5 days of incubation. **(C)** EXO70B1 interacts with AvrPtoB in a GST-pull-down assay. *E. coli*-expressed GST, GST-AvrPtoB, GST-AvrPtoB^F479A^, and GST-AvrPtoB~1--410~ were incubated with plant-expressed EXO70B1-GFP in pull-down assays. After incubation for 4 h at 4°C, the beads were washed three times, and 5 × SDS sample buffer was added. Then, 20 μl of the samples was utilized to detect the precipitated EXO70B1-GFP by anti-GFP antibody, and 10 μl of the samples was used to detect the levels of GST, GST-AvrPtoB, GST-AvrPtoB^F479A^, and GST-AvrPtoB~1--410~ by anti-GST antibody. Arrows indicate the positions of the corresponding proteins. **(D)** EXO70B1 interacts with AvrPtoB in a Co-IP assay. EXO70B1-GFP was co-expressed with AvrPtoB-Myc in *N. benthamiana* leaves. After 48 h, leaves were treated with or without 50 μM of MG132 for 3 h. Total protein was extracted and subjected to immunoprecipitation of AvrPtoB by anti-Myc antibody. Proteins were analyzed in an immunoblot using anti-GFP and anti-Myc antibodies. These experiments were repeated three times with similar results.](fpls-10-01027-g002){#f2}

Previously, we showed that EXO70B1 associates with TN2, and CPK5 phosphorylates EXO70B1 ([@B67]; [@B32]). To examine whether AvrPtoB also interacts with TN2 or CPK5, we introduced constructs encoding AvrPtoB, AvrPtoB~1--410~, and AvrPtoB^F479A^ into yeast along with TN2 or CPK5, with Pto as a positive control. None of these constructs interacted with TN2 or CPK5 ([**Supplementary Figure S1B**](#SM1){ref-type="supplementary-material"}). Taken together, these data indicated that AvrPtoB interacts with EXO70B1, but not TN2 or CPK5, *via* its N-terminal region, and the E3 ligase domain of AvrPtoB is dispensable for the interaction.

To identify the specific domains of AvrPtoB that interact with EXO70B1 and to further confirm that there is no interaction between AvrPtoB and TN2, we generated a series of AvrPtoB truncations, including AvrPtoB~1--205~, AvrPtoB~1--307~, AvrPtoB~1--387~, and AvrPtoB~308--553~ ([**Supplementary Figure S1A**](#SM1){ref-type="supplementary-material"}). Two truncated proteins, AvrPtoB~1--307~ and AvrPtoB~1--387~, interacted with EXO70B1, and none of the deletion mutants interacted with TN2 ([**Figure 2B**](#f2){ref-type="fig"}). As a positive control, Pto did interact with AvrPtoB~1--205~, which indicated that amino acid residues 1--307 of AvrPtoB are sufficient for the interaction between AvrPtoB and EXO70B1.

To test biochemically whether AvrPtoB interacts with EXO70B1, we performed GST-pull-down assays. The *35S:EXO70B1-green fluorescent protein* (*GFP*) vector was transiently expressed in *Nicotiana benthamiana* leaves by *Agrobacterium tumefaciens*-mediated infiltration. Total leaf proteins were extracted, and EXO70B1-GFP was detected by immunoblotting using anti-GFP antibody. Bacterially expressed GST, GST-AvrPtoB, GST-AvrPtoB^F479A^, and GST-AvrPtoB~1--410~ recombinant proteins were incubated with total proteins prepared from *N. benthamiana* leaves transiently expressing EXO70B1-GFP. GST protein was used as the negative control. GST-AvrPtoB, GST-AvrPtoB^F479A^, and GST-AvrPtoB~1--410~ could pull down EXO70B1-GFP, and the EXO70B1-GFP precipitated by GST-AvrPtoB^F479A^ and GST-AvrPtoB~1--410~ is increased possibly because of the degradation activity of wild-type AvrPtoB ([**Figure 2C**](#f2){ref-type="fig"}).

To further test the interaction between AvrPtoB and EXO70B1 *in planta*, we performed Co-IP assays by transiently expressing EXO70B1-GFP and AvrPtoB-Myc in *N. benthamiana* leaves, using EXO70B1-GFP alone as a negative control. AvrPtoB was immunoprecipitated using anti-Myc antibody, and EXO70B1 was detected with anti-GFP antibody only in the precipitate from the leaves that co-expressed both EXO70B1-GFP and AvrPtoB-Myc, but not from the negative control leaves that only expressed EXO70B1-GFP. Interestingly, the protein level of EXO70B1 and the association of EXO70B1 and AvrPtoB were markedly increased upon treatment with MG132, an inhibitor of the proteasome complex ([@B5]) ([**Figure 2D**](#f2){ref-type="fig"}). These results indicated that EXO70B1 and AvrPtoB form a protein complex in *N. benthamiana*, and AvrPtoB may mediate the degradation of EXO70B1.

AvrPtoB Degrades EXO70B1 in a Proteasome-Dependent Manner {#s3_2}
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The interaction between EXO70B1 and AvrPtoB suggests that EXO70B1 may be a substrate of AvrPtoB and may be degraded by AvrPtoB. To investigate this possibility, we first conducted a cell-free degradation assay. The *35S:AvrPtoB-Myc* or an empty vector control construct was transiently expressed in *N. benthamiana* leaves. Total leaf proteins were extracted, and AvrPtoB-Myc was identified by immunoblotting using anti-Myc antibody. Bacterially expressed MBP or MBP-EXO70B1 recombinant proteins were incubated with total proteins prepared from *N. benthamiana* leaves transiently expressing AvrPtoB-Myc or Myc with or without MG132 and were subjected to immunoblot analysis with anti-MBP antibody at the indicated time. After 1.5 h of incubation, the level of MBP-EXO70B1 was slightly reduced in the AvrPtoB-Myc cell-free extracts. After 3 h of incubation, the level of MBP-EXO70B1 was substantially reduced in the AvrPtoB-Myc cell-free extracts. By contrast, MBP-EXO70B1 remained stable in the Myc cell-free extracts and in the presence of MG132 ([**Figure 3A**](#f3){ref-type="fig"}), indicating that MBP-EXO70B1 is degraded in a proteasome-dependent manner in the presence of AvrPtoB-Myc. The MBP protein, as a negative control, remained unaffected in both extracts.

![The degradation and ubiquitination of EXO70B1 by AvrPtoB. **(A)** Cell-free degradation assay of EXO70B1 by AvrPtoB. Bacterially expressed MBP or MBP-EXO70B1 was incubated with a cell-free crude extract prepared from *N. benthamiana* leaves transiently expressing AvrPtoB-Myc or Myc, in the presence or absence of 50 μM of MG132. The time-dependent changes of protein levels were monitored by immunoblotting with anti-MBP antibody. Numbers indicate the protein levels of MBP-EXO70B1 normalized to MBP using ImageJ software. The levels of Rubisco are shown as an equal loading control of cell-free extracts. **(B)** *In vivo* degradation assay of EXO70B1 by AvrPtoB in protoplasts. The *35S:EXO70B1-HA* and *35S:GFP* constructs were cotransfected with the *35S:Myc*, *35S:AvrPtoB-Myc*, or *35S:AvrPtoB^F479A^-Myc* constructs into protoplasts freshly isolated from wild-type *Arabidopsis* leaf tissues using PEG-mediated transformation. After overnight transfection, protoplasts were incubated with or without 50 μM of MG132 for 3 h. Total proteins were extracted and examined by immunoblot analysis with anti-HA, anti-GFP, and anti-Myc antibodies. The levels of GFP show the transfection efficiency of protoplasts. Numbers indicate the protein levels of EXO70B1-HA normalized to GFP using ImageJ software. The levels of Rubisco are shown as an equal loading control. **(C)** EXO70B1-GFP and AvrPtoB-Myc levels in the indicated genotypes were determined by immunoblotting using anti-GFP and anti-Myc antibodies. Ponceau S staining of Rubisco is shown as a protein loading control. **(D)** RT-qPCR analysis of *EXO70B1* expression. Total RNA was isolated from leaves of the 5-week-old *Arabidopsis* plants. The expression levels of *EXO70B1* were normalized to the reference gene *ACTIN2*. Data represent the mean and standard deviation of three biological replicates. Three technical replicates for each biological sample were used. The lowercase letters indicate statistically significant differences (*P* \< 0.05; one-way ANOVA). **(E)** *In vitro* self-ubiquitination assay of AvrPtoB. Recombinant GST-AvrPtoB was incubated in ubiquitination buffer in the presence or absence of ubiquitin, E1 (UBE1), or E2 (UBC8) at 30°C for 2 h. Recombinant GST-AvrPtoB^F479A^ was used as a negative control. Reaction products were resolved using SDS-PAGE and subjected to immunoblot analysis with anti-GST and anti-Ub antibodies. Asterisk indicates the contaminating bands. **(F)** *In vitro* ubiquitination assay of EXO70B1 by AvrPtoB. Recombinant MBP-EXO70B1 was incubated in ubiquitination buffer in the presence or absence of ubiquitin, E1 (UBE1), E2 (UBC8), or E3 (AvrPtoB or AvrPtoB^F479A^). The reaction mixtures were subjected to immunoblot analysis with anti-MBP and anti-GST antibodies. Ubiquitinated MBP-EXO70B1 was detected by anti-MBP antibody. These experiments were repeated three times with similar results.](fpls-10-01027-g003){#f3}

To further confirm that AvrPtoB mediates the degradation of EXO70B1, we next performed a protoplast transient expression assay. The EXO70B1-HA and GFP proteins were transiently co-expressed with AvrPtoB-Myc or AvrPtoB^F479A^-Myc in protoplasts prepared from *Arabidopsis* Col-0 leaves. After overnight transfection, protoplasts were incubated with or without MG132 for another 3 h. Total proteins were extracted and examined by immunoblotting analysis with anti-HA, anti-GFP, and anti-Myc antibodies. The level of EXO70B1-HA was decreased in the presence of AvrPtoB-Myc, but not AvrPtoB^F479A^-Myc ([**Figure 3B**](#f3){ref-type="fig"}). The amount of EXO70B1-HA was increased in the presence of MG132 ([**Figure 3B**](#f3){ref-type="fig"}), suggesting that the degradation of EXO70B1-HA by AvrPtoB-Myc *in vivo* in an E3-ligase-dependent manner is controlled by the 26S proteasome. The level of GFP, which was used to assess the efficiency of protoplast transfection, and Rubisco, which was shown as an equal protein loading control, remained unchanged regardless of the presence of MG132. Taken together, these data indicated that the degradation of EXO70B1 is regulated by AvrPtoB in a proteasome-dependent manner.

To further analyze whether AvrPtoB regulates the degradation of EXO70B1 in *Arabidopsis*, we introduced the *35S:AvrPtoB-Myc* clone into the *exo70B1-3* transgenic line, which contains a construct encoding functional EXO70B1-GFP in the *exo70B1-3* mutant background ([@B67]). Several independent stable transgenic lines were obtained, and the *35S:AvrPtoB-Myc* \#2 and \#7 plants were chosen for further research. We extracted total proteins from 5-week-old Col-0, the *exo70B1-3* transgenic line, and the *35S:AvrPtoB-Myc* \#2 and \#7 plants and detected EXO70B1-GFP and AvrPtoB-Myc by immunoblotting analysis with anti-GFP and anti-Myc antibodies, respectively. The *35S:AvrPtoB-Myc* \#2 and \#7 transgenic plants expressed AvrPtoB-Myc of the correct size, and the amount of EXO70B1-GFP was lower in the *35S:AvrPtoB-Myc* transgenic plants compared with the controls ([**Figure 3C**](#f3){ref-type="fig"}). We also tested the transcript levels of *EXO70B1* and found that they were slightly increased in the *35S:AvrPtoB-Myc* \#2 and \#7 plants ([**Figure 3D**](#f3){ref-type="fig"}), indicating that AvrPtoB destabilizes EXO70B1 by post-translational regulation.

To investigate whether EXO70B1 is a substrate of AvrPtoB, we performed *in vitro* ubiquitination assays to analyze whether AvrPtoB can ubiquitinate EXO70B1. First, we used human E1 (UBE1) and *Arabidopsis* E2 (UBC8) to assess the *in vitro* E3 ligase activity of AvrPtoB. Recombinant full-length GST-AvrPtoB and GST-AvrPtoB^F479A^ were incubated with ubiquitination buffer in the presence or absence of ubiquitin (Ub), UBE1, and UBC8 at 30°C for 2 h. The reaction mixture was analyzed by immunoblotting using anti-Ub and anti-GST antibodies. We observed GST-AvrPtoB E3 ligase activity as evidenced by high-molecular-mass smear ladders detected by both anti-Ub and anti-GST antibodies ([**Figure 3E**](#f3){ref-type="fig"}), indicating that GST-AvrPtoB could self-ubiquitinate in our experimental conditions. In contrast, the E3-ligase-deficient mutation of AvrPtoB, GST-AvrPtoB^F479A^, did not have E3 ligase activity ([**Figure 3E**](#f3){ref-type="fig"}).

Next, a bacterially expressed MBP-EXO70B1 recombinant protein was used for *in vitro* ubiquitination assays in the presence of all the other components required for the ubiquitination reaction. After 3 h of incubation, multiple ubiquitinated forms of MBP-EXO70B1 appeared in the presence of Ub, UBE1, UBC8, and GST-AvrPtoB. However, coincubation of MBP-EXO70B1 with GST-AvrPtoB^F479A^ failed to produce any detectable high-molecular-mass smear ladders ([**Figure 3F**](#f3){ref-type="fig"}), suggesting that the presence of an active E3 ligase domain is required for the ubiquitination of MBP-EXO70B1 by GST-AvrPtoB. Taken together, these data indicated that EXO70B1 is a substrate of AvrPtoB and is ubiquitinated by AvrPtoB.

Since EXO70B1 and EXO70B2 show the highest sequence similarity and both are required for PTI responses ([@B48]), we also analyzed whether AvrPtoB targets EXO70B2. With Pto as a positive control, we found that EXO70B2 also interacted with AvrPtoB ([**Supplementary Figure S2A**](#SM1){ref-type="supplementary-material"}). Next, with MBP-EXO70B1 as a positive control, no ubiquitination form of MBP-EXO70B2 was detected in the presence of all required ubiquitination components in our experimental condition ([**Supplementary Figure S2B**](#SM1){ref-type="supplementary-material"}). Taken together, these data indicated that EXO70B1, not EXO70B2, is a substrate of AvrPtoB.

Overexpression of AvrPtoB Leads to Stunted Phenotypes in *Arabidopsis* {#s3_3}
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Further on, we analyzed the phenotypes of transgenic *Arabidopsis* plants that consistently express AvrPtoB. Compared with Col-0 and the *exo70B1-3* transgenic line, 5-week-old *35S:AvrPtoB-Myc* \#2 and \#7 transgenic plants displayed stunted phenotypes and growth defects ([**Figure 4A**](#f4){ref-type="fig"}), which were similar to those in TN2 overexpressing plants ([@B31]; [@B36]). However, no obvious spontaneous cell death was observed in the *35S:AvrPtoB-Myc* plants.

![Overexpression of AvrPtoB leads to stunted phenotypes in *Arabidopsis*. **(A)** Five-week-old plants of Col-0, the *exo70B1-3* transgenic line (expressing EXO70B1-GFP in the *exo70B1-3* mutant background), and two independent *35S:AvrPtoB-Myc* transgenic *Arabidopsis* plants in the background of the *exo70B1-3* transgenic line were photographed under short-day conditions. Bar = 1.0 cm. **(B--E)** RT-qPCR analysis of *TN2* **(B)**, *PR1* **(C)**, *PR2* **(D)**, and immune-related genes **(E)** expression. Total RNA was isolated from leaves of the 5-week-old *Arabidopsis* plants indicated in **(A)**. The expression levels of indicated gene were normalized to the reference gene *ACTIN2*. Data represent the mean and standard deviation of three biological replicates. Three technical replicates for each biological sample were used. The lowercase letters indicate statistically significant differences (*P* \< 0.05; one-way ANOVA). These experiments were repeated three times with similar results.](fpls-10-01027-g004){#f4}

Next, we analyzed the transcript levels of *TN2* in 5-week-old Col-0, the *exo70B1-3* transgenic line, and the *35S:AvrPtoB-Myc* \#2 and \#7 plants and found that the expression of *TN2* was significantly up-regulated in the *35S:AvrPtoB-Myc* \#2 and \#7 plants compared with the wild type and the *exo70B1-3* transgenic line ([**Figure 4B**](#f4){ref-type="fig"}). In *exo70B1-3* mutant, the expression of *PR1* and *PR2* is constitutively up-regulated due to the activated TN2 ([@B67]; [@B32]). Similarly, we found that the expression levels of *PR1* and *PR2* were also significantly up-regulated in the *35S:AvrPtoB-Myc* \#2 and \#7 plants ([**Figures 4C, D**](#f4){ref-type="fig"}). We further examined the expression of other immune-related genes, including *PHYTOALEXIN DEFICIENT 4* (*PAD4*), *NPR1*, and *ENHANCED DISEASE SUSCEPTIBILITY 5* (*EDS5*). The transcript levels of these genes were significantly up-regulated in the *35S:AvrPtoB-Myc* \#2 and \#7 plants ([**Figure 4E**](#f4){ref-type="fig"}). As shown in [**Figure 3C**](#f3){ref-type="fig"}, AvrPtoB-Myc \#7 accumulated much higher AvrPtoB-Myc protein levels and lower EXO70B1-GFP protein levels than did AvrPtoB-Myc \#2, and the transcript levels of immune-related genes in AvrPtoB-Myc \#7 were higher than those in AvrPtoB-Myc \#2, which indicated that the stunted phenotypes and the up-regulation of immune-related genes in AvrPtoB-Myc plants are related to the low EXO70B1 protein levels. Taken together, these data indicated that overexpression of AvrPtoB leads to low accumulation of EXO70B1 and autoimmunity phenotypes, which may be caused by TN2-triggered plant immunity.

The Autoimmunity Phenotypes in the AvrPtoB Overexpression Lines Are Partially Dependent on TN2 {#s3_4}
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To investigate whether there is a direct link between the phenotypes of *35S:AvrPtoB-Myc* plants and the accumulation of TN2, we crossed the *35S:AvrPtoB-Myc* \#2 and \#7 plants with the *tn2-10* mutant, a T-DNA insertion mutant of *TN2*. The *tn2-10* mutation fully suppressed the spontaneous cell death and powdery mildew resistance in the *exo70B1-3* mutant, indicating that *tn2-10* is a loss-of-function mutation ([**Supplementary Figures S3A--C**](#SM1){ref-type="supplementary-material"}). The expression level of *TN2* was up-regulated in the *exo70B1-3* mutants, which is consistent with our early reports ([@B67]; [@B32]), and was not detected in *tn2-10* mutants and *exo70B1-3 tn2-10* double mutants ([**Supplementary Figure S3D**](#SM1){ref-type="supplementary-material"}). We obtained stable *35S:AvrPtoB-Myc* transgenic plants in the Col-0 (*35S:AvrPtoB-Myc* \#2/Col-0 and *35S:AvrPtoB-Myc* \#7/Col-0) and *tn2-10* mutant (*35S:AvrPtoB-Myc* \#2/*tn2-10* and *35S:AvrPtoB-Myc* \#7/*tn2-10*) background by crossing. We observed the stunted phenotypes of the *35S:AvrPtoB-Myc* \#2 and \#7 plants in the Col-0 background, which was indistinguishable from the phenotypes of the *35S:AvrPtoB-Myc* \#2 and \#7 plants in the background of the *exo70B1-3* transgenic line. In contrast, the stunted phenotypes were partially rescued by the *tn2-10* mutation ([**Figure 5A**](#f5){ref-type="fig"}). The levels of AvrPtoB-Myc were similar in the *35S:AvrPtoB-Myc* \#2 and \#7 plants in the Col-0 and *tn2-10* background ([**Figure 5B**](#f5){ref-type="fig"}), indicating that the stunted phenotype caused by the expression of AvrPtoB-Myc is partially dependent on TN2.

![The stunted phenotypes of the AvrPtoB overexpression lines are partially dependent on TN2. **(A)** Five-week-old Col-0, *tn2-10*, and two independent *35S:AvrPtoB-Myc* transgenic *Arabidopsis* plants (*35S:AvrPtoB-Myc* \#2 and *35S:AvrPtoB-Myc* \#7) in the Col-0 and *tn2-10* background were photographed under short-day conditions. Bar = 1.0 cm. **(B)** The AvrPtoB-Myc fusion protein was examined by immunoblotting. Ponceau S staining of Rubisco was shown as a protein loading control. **(C--H)** RT-qPCR analysis of *TN2* **(C)**, *PR1* **(D)**, *PR2* **(E)**, *PAD4* **(F)**, *NPR1* **(G)**, and *EDS5* **(H)** expression. Total RNA was isolated from leaves of the indicated 5-week-old *Arabidopsis* plants. The expression levels of indicated gene were normalized to the reference gene *ACTIN2*. Data represent the mean and standard deviation of three biological replicates. Three technical replicates for each biological sample were used. The lowercase letters indicate statistically significant differences (*P* \< 0.05; one-way ANOVA). These experiments were repeated three times with similar results.](fpls-10-01027-g005){#f5}

Consistent with the phenotypes, the transcript levels of *TN2* were up-regulated in *35S:AvrPtoB-Myc* \#2/Col-0 and *35S:AvrPtoB-Myc* \#7/Col-0 plants ([**Figure 5C**](#f5){ref-type="fig"}), and the up-regulation of *PR1*, *PR2*, *PAD4*, *NPR1*, and *EDS5* expression levels was partially suppressed by the *tn2-10* mutation ([**Figures 5D--H**](#f5){ref-type="fig"}). Taken together, these data indicated that overexpression of AvrPtoB in *Arabidopsis* causes a decrease of EXO70B1 and activates TN2-triggered immunity.

AvrPtoB Overcomes EXO70B1-Mediated Resistance {#s3_5}
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Previously, we showed that *TN2* transcripts were up-regulated only in mature *exo70B1-3* plants ([@B67]), and the expression level of *TN2* in 10-day-old *exo70B1-3* seedlings was similar to that in Col-0 ([**Figure 6A**](#f6){ref-type="fig"}). However, why the *TN2* mRNA level did not change in young seedlings of *exo70B1-3* mutant is still unclear. As the *TN2* mRNA level did not change in young seedlings of *exo70B1-3* mutant, the phenotypes of young *exo70B1-3* seedlings would reflect the real role of EXO70B1 in resistance. Therefore, to assess whether degradation of EXO70B1 by AvrPtoB is important for the proliferation of *Pto* DC3000 in its host, we examined 10-day-old *exo70B1-3* seedlings for responses to *Pto* DC3000 and the *Pto* DC3000 mutant strain lacking *AvrPtoB* (*Pto ΔAvrPtoB*). The virulence of the *Pto ΔAvrPtoB* strain was strongly reduced in Col-0 ([**Figure 6B**](#f6){ref-type="fig"}), which is consistent with earlier reports ([@B24]; [@B19]). In the *exo70B1-3* mutant seedlings, bacterial numbers increased to similar extents for both *Pto* DC3000 strains, indicating that EXO70B1 contributes to bacterial resistance in *Arabidopsis*, and AvrPtoB enhances the virulence of *Pto* DC3000 by overcoming EXO70B1-mediated resistance.

![AvrPtoB overcomes EXO70B1-mediated resistance. **(A)** *TN2* transcript levels were examined by RT-qPCR. Total RNA was isolated from the indicated 10-day-old seedlings. The mRNA expression level was normalized to the reference gene *ACTIN2*. Data represent the mean and standard deviation of three biological replicates. Three technical replicates for each biological sample were used. This experiment was repeated three times with similar results. **(B)** AvrPtoB contributes to the virulence of *Pto* DC3000, which is dependent on its ability to disable the function of EXO70B1. Ten-day-old Col-0 and *exo70B1-3* seedlings were infected with *Pto* DC3000 and *Pto ΔAvrPtoB*. The bacterial growth assays were performed 3 days after infection. Results represent the mean and standard deviation of three independent experiments each consisting four independent biological samples. cfu, colony-forming units. The lowercase letters indicate statistically significant differences (*P* \< 0.05; nested ANOVA). **(C)** and **(D)** *Pto* DC3000 enhances the expression of *EXO70B1*. Leaves of soil-grown wild-type Col-0 *Arabidopsis* plants were hand infiltrated with *Pto* DC3000 or the *Pto ΔAvrPtoB* mutant at 10^8^ cfu/ml and harvested at 24-h postinoculation. The transcript levels of *PR1* and *EXO70B1* were measured by RT-qPCR as described above. Data represent the mean and standard deviation of three biological replicates. Three technical replicates for each biological sample were used. These experiments were repeated three times with similar results. The lowercase letters indicate statistically significant differences (*P* \< 0.05; one-way ANOVA).](fpls-10-01027-g006){#f6}

To gain more insight into the function of EXO70B1 in plant immunity, we analyzed the transcripts of *EXO70B1* as well as *PR1* in Col-0 plants after inoculation with *Pto* DC3000 and *Pto ΔAvrPtoB*. Similar to the recent report, the *PR1* transcript level was lower in plants inoculated with *Pto* DC3000 than with *Pto ΔAvrPtoB* ([@B6]) ([**Figure 6C**](#f6){ref-type="fig"}). The expression levels of *EXO70B1* were up-regulated after inoculation with both strains, and the up-regulation was enhanced in the presence of AvrPtoB ([**Figure 6D**](#f6){ref-type="fig"}).

EXO70B1 Suppresses TN2-Triggered Cell Death in Tobacco {#s3_6}
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To further investigate the relationship between EXO70B1 and TN2, we expressed these proteins in *N. tabacum*. The expression of TN2 or TN2-TIR triggered a strong and rapid hypersensitive response (HR). This TN2- or TN2-TIR-triggered cell death was suppressed by co-expression with EXO70B1, not EXO70B2 ([**Figures 7A, B**](#f7){ref-type="fig"}). TN2- or TN2-TIR-triggered cell death was also suppressed when we co-expressed TN2 or TN2-TIR with EXO70B1 using different *Agrobacterium* GV3101 concentrations, and proteins of the correct size were detected by immunoblotting analysis ([**Supplementary Figure S4**](#SM1){ref-type="supplementary-material"}). These results indicated that EXO70B1 may maintain TN2 in an inactive status, and TN2 triggers cell death only in the absence of EXO70B1.

![EXO70B1 suppresses TN2- or TN2-TIR-triggered cell death in *N. tabacum*. **(A)** and**(B)** TN2- or TN2-TIR-triggered cell death was suppressed by EXO70B1, but not EXO70B2, in *N. tabacum*. **(C)** and **(D)** AvrPtoB rescues TN2- or TN2-TIR-triggered cell death, which is suppressed by EXO70B1, in *N. tabacum*. *Agrobacterium* GV3101 cells carrying different constructs were mixed prior to adding infiltration buffer. The concentration of each agrobacteria in the mix was brought to OD~600~ = 0.5 and infiltrated into 5-week-old *N. tabacum* leaves for transient expression. Cell death was observed 36 to 48 h later. Pictures were taken 4 days postinfiltration. These experiments were repeated three times with similar results.](fpls-10-01027-g007){#f7}

To further study the role of AvrPtoB in TN2-triggered cell death, we co-expressed TN2 or TN2-TIR with EXO70B1 and AvrPtoB in *N. tabacum*. As shown in [**Figures 7C, D**](#f7){ref-type="fig"}, AvrPtoB rescued TN2- or TN2-TIR-triggered cell death, which was suppressed by EXO70B1. Similar results were observed in *N. benthamiana* leaves ([**Supplementary Figure S5**](#SM1){ref-type="supplementary-material"}). Taken together, these data indicated that EXO70B1 associates with TN2 and suppresses the activation of TN2, and TN2 is activated after the degradation of EXO70B1 by AvrPtoB in both *N. tabacum* and *N. benthamiana*.

Discussion {#s4}
==========

EXO70B1 is a component of the exocyst complex and belongs to the EXO70 protein family. In this study, we show that EXO70B1 is required for early immune signaling events, such as the ROS burst and callose deposition, triggered by flg22, elf18, and chitin ([**Figure 1**](#f1){ref-type="fig"}), which is consistent with the previous report on the requirement for EXO70B1 in flg22-triggered ROS burst ([@B48]). We also show that AvrPtoB, an effector of *Pto* DC3000, targets EXO70B1 to suppress plant immunity ([**Figures 2**](#f2){ref-type="fig"}, [**3**](#f3){ref-type="fig"}, and [**6**](#f6){ref-type="fig"}). Overexpression of AvrPtoB in *Arabidopsis* leads to autoimmunity, which is partially dependent of TN2 ([**Figures 4**](#f4){ref-type="fig"} and [**5**](#f5){ref-type="fig"}). In addition, EXO70B1 inhibits the cell death triggered by TN2 ([**Figure 7**](#f7){ref-type="fig"} and [**Supplementary Figure S4**](#SM1){ref-type="supplementary-material"}), suggesting that EXO70B1 associates with TN2 and may maintain TN2 in an inactive status.

In vesicle trafficking, exocytosis plays fundamental roles by maintaining membrane integrity and contributing to membrane remodeling in response to altered environmental conditions ([@B13]). RIN4 recruits EXO70B1 to the PM, and AvrRpt2 can release both RIN4 and EXO70B1 to the cytoplasm, indicating that the localization of EXO70B1 at PM may be required for plant immunity ([@B44]). The *exo70B1-3* mutant was compromised in early responses to multiple PAMPs, which is likely due to impaired PRRs signaling. Future studies will need to determine whether EXO70B1-associated exocyst complex contributes to the recycling of important components in PTI signaling. Despite the importance of the exocyst complex in vesicle trafficking and immune signaling, very few effectors are reported to target the components of the exocyst complex. In rice, the effector Avr-Pii from the rice blast fungus Magnaporthe oryzae interacts with OsEXO70F2 and OsEXO70F3, and OsEXO70F3, not EXO70F2, is specifically involved in Pii-dependent resistance ([@B17]). Similarly, in this study, we show that both EXO70B1 and EXO70B2 interact with AvrPtoB ([**Figure 2**](#f2){ref-type="fig"} and [**Supplementary Figure S2**](#SM1){ref-type="supplementary-material"}), but only EXO70B1 is ubiquitinated by AvrPtoB ([**Figure 3**](#f3){ref-type="fig"} and [**Supplementary Figure S2**](#SM1){ref-type="supplementary-material"}). These results indicated that AvrPtoB associates with both EXO70B1 and EXO70B2 as EXO70B1 and EXO70B2 show the highest sequence similarity, but AvrPtoB specifically ubiquitinates and mediates the degradation of EXO70B1 to suppress EXO70B1-related plant immune responses.

The amino acid residues 206--307 of AvrPtoB, also known as the NPR1-interacting domain (NID) ([@B6]), are necessary for the interaction between AvrPtoB and EXO70B1 ([**Figure 2D**](#f2){ref-type="fig"}). This domain is sufficient for the interaction between AvrPtoB and most of its targets, including NPR1, MKK2 (MAP kinase kinase 2), Bti9 (AvrPtoB tomato-interacting protein 9), and CERK1 ([@B18]; [@B63]; [@B55]; [@B6]). Besides the NID, the Fen-interacting domain (FID) of AvrPtoB, encoded by amino acid residues 308--387, is another important region for the interaction between AvrPtoB and its targets, including Fen, FLS2, and BAK1 ([@B41]; [@B19]). Among the AvrPtoB-targeted proteins, only Pto binds to the Pto-interacting domain (PID) between amino acids 121 and 200, and to the FID domain of AvrPtoB, all other targets of AvrPtoB bind to the 206--387 amino acid region (NID and FID) of AvrPtoB. However, no interaction was observed between the full-length AvrPtoB and EXO70B1 in Y2H assay maybe because of the AvrPtoB E3 ligase activity. It is likely in the early report that the E3 ligase-deficient AvrPtoB mutants, not wild-type AvrPtoB, interacted with Fen in Y2H assay ([@B41]). Further study is also needed to investigate why the N-terminal region of AvrPtoB can target so many different proteins, including protein kinases, transcriptional coactivator NPR1, and the subunit of exocyst complex EXO70B1.

AvrPtoB targets a number of regulators of plant immunity and contributes to the virulence of *Pto* DC3000. However, there is no evidence that any targets of AvrPtoB are monitored by NLR proteins and trigger ETI in *Arabidopsis* when targeted. Here, we showed that AvrPtoB ubiquitinates and degrades EXO70B1, and overexpression of AvrPtoB in *Arabidopsis* leads to autoimmunity, which is similar to the phenotype of TN2 overexpression plants reported previously ([@B31]; [@B36]), but not to the phenotype of *exo70B1* mutants ([**Figure 4**](#f4){ref-type="fig"}). The most like reason is that the *TN2* transcripts were only up-regulated in older *exo70B1-3* plants ([@B67]), which is different from *TN2* overexpression plants. Therefore, AvrPtoB overexpression phenotypes maybe due to the consistent up-regulation of TN2. Moreover, the autoimmunity phenotypes of the *35S:AvrPtoB-Myc* transgenic plants are partially dependent on TN2 ([**Figure 5**](#f5){ref-type="fig"}). The reason why the *tn2-10* mutation does not fully rescue the stunted phenotypes of the *35S:AvrPtoB-Myc* transgenic plants may be because there are many targets of AvrPtoB in *Arabidopsis* and AvrPtoB may trigger other forms of NLR-mediated resistance. For instance, AvrPtoB also targets BAK1, and loss-of-function *bak1* mutants show a cell death phenotype ([@B28]), which could be caused by activation of an undefined NLR protein.

*Pto* DC3000, a bacterial strain carrying AvrPtoB, did not show a reduced fitness postinoculation to Col-0 ([**Figure 6B**](#f6){ref-type="fig"}). The most likely explanation for this is that AvrPtoB targets lots of proteins functioning in plant immunity, and modifications of other targets by AvrPtoB may contribute to its virulence, which overcome TN2-mediated resistance. For instance, NPR1, which contributes to TN2-mediated resistance ([@B67]), is also targeted by AvrPtoB ([@B6]), and the modification of NPR1 by AvrPtoB could interfere with TN2-mediated resistance. In addition, the amount of AvrPtoB secreted into plant cells by *Pto* DC3000 is limited, which is insufficient to trigger TN2-mediated resistance. Furthermore, several other effectors can interact with EXO70B1, and those effectors may modify EXO70B1 as well. The modification of EXO70B1 by other effectors may influence the ubiquitination and degradation of EXO70B1 mediated by AvrPtoB. Therefore, TN2-mediated resistance is not activated.

Ectopic expression of the full-length or truncated TIR domain of some NLR proteins in the absence of pathogens can trigger an HR in *N. tabacum* ([@B51]; [@B4]). In this study, we show that transient expression of TN2 or TN2-TIR alone triggers strong and rapid cell death in *N. tabacum*, which is suppressed by co-expression of EXO70B1, not EXO70B2 ([**Figure 7**](#f7){ref-type="fig"}), indicating that EXO70B1 specifically suppresses the activation of TN2, and which is consistent with results on TN2 interaction with EXO70B1, but not with EXO70B2 ([**Supplementary Figure S2A**](#SM1){ref-type="supplementary-material"}). Additionally, TN2 and TN2-TIR accumulated in the presence of EXO70B1 ([**Supplementary Figure S4**](#SM1){ref-type="supplementary-material"}), indicating that EXO70B1 associates with TN2 and may maintain TN2 in an inactive status, rather than to degrade TN2 by EXO70B1-associated autophagy activity.

The genome of the *Arabidopsis* ecotype Col-0 encodes about 50 atypical NLR proteins, including TIR, TIR-X, and TIR-NBS ([@B35]), some of which have been reported to play roles in plant immunity. For instance, RESPONSE TO HOPBA1 (RBA1), a TIR-only protein, functions as a pathogen sensor and recognizes the HopBA1 effector directly ([@B38]). TN13 is a MODIFIER OF SNC1, 6 (MOS6) interaction partner required for resistance against *Pto* DC3000 lacking AvrPto and AvrPtoB ([@B42]). However, how truncated NLR proteins activate disease resistance is not clear. It is proposed that the truncated NLR proteins might not function alone and instead play a role in immunity with full-length NLR proteins ([@B36]). Future studies are needed to determine whether overexpression of AvrPtoB triggered autoimmunity and TN2-activated resistance rely on full-length NLR proteins.

Conclusion {#s5}
==========

Our study demonstrated that EXO70B1, a subunit of the exocyst complex, plays an important role in immune responses triggered by multiple PAMPs. *Pto* DC3000 secretes AvrPtoB into plant cells to target EXO70B1, which may contribute to the trafficking of plasma membrane proteins involved in PAMP-triggered immunity. EXO70B1 associates with and maintains TN2, a truncated NLR protein, in an inactive status. Although the role of EXO70B1 in plant immunity and how EXO70B1 inhibits TN2 activation need further study, our results highlight that the *Arabidopsis* exocyst component is a target of effectors and may be guarded by a truncated NLR protein.

Data Availability {#s6}
=================

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: AT5G03540 (At EXO70A1), At5g58430 (At EXO70B1), At1g07000 (At EXO70B2), At1g17615 (At TN2), At2g14610 (At PR1), AT3g57260 (At PR2), At3g52430 (At PAD4), At1g64280 (At NPR1), At4g39030 (At EDS5), At3g18780 (At ACTIN2), AAF76306 (Sl Pto), AAO57459 (AvrPto), AY074795 (AvrPtoB), AAO54130 (Hop H1), AAO53599 (Hop K1), AF458392 (Hop O1-1), and AF458403 (Hop Y1).

Author Contributions {#s7}
====================

DT and WW conceived and initiated the research and designed the experiments; WW, NL, CG, and LR performed the experiments; WW, NL, CG, and DT analyzed the data; DT and WW wrote the paper.

Funding {#s8}
=======

The work was supported by grants from the National Natural Science Foundation of China (31761133017) and National Science Fund for Distinguished Young Scholars of China (31525019) to DT.

Conflict of Interest Statement {#s9}
==============================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We thank Dr. Jian-Min Zhou for *Pto ΔAvrPtoB* strain, Dr. Gitta Coaker for *N. tabacum* seeds, Dr. Shuhua Yang for pSuper1300 vectors, and Dr. Qi Xie for anti-Ub antibody.

Supplementary Material {#s10}
======================

The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fpls.2019.01027/full#supplementary-material>

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[^1]: Edited by: Aardra Kachroo, University of Kentucky, United States

[^2]: Reviewed by: Seong Wook Yang, Yonsei University, South Korea; Tamara Pecenkova, Institute of Experimental Botany (ASCR), Czechia

[^3]: This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Plant Science