NRG1 functions downstream of EDS1 to regulate TIR-NLR-mediated plant immunity in
            <i>Nicotiana benthamiana</i>

Qi, Tiancong; Seong, Kyungyong; Thomazella, Daniela P. T.; Kim, Joonyoung Ryan; Pham, Julie; Seo, Eun-Young; Cho, Myeong‐Je; Schultink, Alex; Staskawicz, Brian J.

doi:10.1073/pnas.1814856115

Cited by 187 publications

(303 citation statements)

References 56 publications

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“…benthamiana NRG1, Qi et al . () indeed observed its oligo‐ or dimerization that relies on the CC domain. Furthermore, association between NRG1 and EDS1 also was detected using N .…”

Section: Discussionmentioning

confidence: 87%

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Differential regulation of TNL‐mediated immune signaling by redundant helper CNLs

et al. 2019

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Summary Higher plants utilize nucleotide‐binding leucine‐rich repeat domain proteins (NLRs) as intracellular immune receptors to recognize pathogen‐derived effectors and trigger a robust defense. The Activated Disease Resistance 1 (ADR1) family of coiled‐coil NLRs (CNLs) have evolved as helper NLRs that function downstream of many TIR‐type sensor NLRs (TNLs). Close homologs of ADR1s form the N REQUIREMENT GENE 1 (NRG1) family in Arabidopsis, the function of which is unclear. Through CRISPR/Cas9 gene editing methods, we discovered that the tandemly repeated NRG1A and NRG1B are functionally redundant and operate downstream of TNLs with differential strengths. Interestingly, ADR1s and NRG1s function in two distinct parallel pathways contributing to TNL‐specific immunity. Synergistic effects on basal and TNL‐mediated defense were detected among ADR1s and NRG1s. An intact P‐loop of NRG1s is not required for mediating signals from sensor TNLs, whereas auto‐active NRG1A exhibits autoimmunity. Importantly, NRG1s localize to the cytosol and endomembrane network regardless of the presence of effectors, suggesting a cytosolic activation mechanism. Taken together, different sensor TNLs differentially use two groups of helper NLRs, ADR1s and NRG1s, to transduce downstream defense signals.

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“…benthamiana NRG1, Qi et al . () indeed observed its oligo‐ or dimerization that relies on the CC domain. Furthermore, association between NRG1 and EDS1 also was detected using N .…”

Section: Discussionmentioning

confidence: 87%

“…Supporting this, a recent study showed the requirement of NRG1 in several TNL‐mediated, but not CNL‐mediated, immunity pathways in N . benthamiana (Qi et al ., ). By contrast, the ADR1 family is conserved among various plant species including monocots and eudicots (Collier et al ., ).…”

Section: Discussionmentioning

confidence: 97%

Differential regulation of TNL‐mediated immune signaling by redundant helper CNLs

et al. 2019

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“…By contrast, as c . 20% of the ETI genes are still normally regulated in nrg1 (Qi et al ., ), SA signalling is probably not the only immune response still active in nrg1 mutants.…”

Section: Discussionmentioning

confidence: 94%

“…By contrast, NRG1 is required for Roq1‐mediated HR and Roq1‐dependent bacterial resistance in N. benthamiana (Qi et al ., ). We speculate that ADR1 proteins, from the sister clade of NRG1, might complement the loss of NRG1s for some TIR‐NLRs, e.g.…”

Section: Discussionmentioning

confidence: 97%

“…We found that NRG1 is at least partially required for the signalling of the TIR‐NLRs WRR4A, WRR4B, RPP1, RPP2 and RPP4, the NLR pairs CHS1/SOC3, CHS3/CSA1 and RRS1/RPS4, and the CC‐NLRs RPS2 and RPM1, but is not required for the signalling of the CC‐NLRs RPS5 and MLA7. Other recent studies have also suggested a requirement of NRG1 for RPP1‐mediated signalling and have identified Roq1, an additional NRG1‐dependent TIR‐NLR (Brendolise et al ., ; Qi et al ., ). Surprisingly, we found that NRG1 is required for RRS1/RPS4‐mediated HR, but not bacterial disease resistance conferred by RRS1/RPS4.…”

Section: Introductionmentioning

confidence: 97%

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Diverse NLR immune receptors activate defence via the RPW8‐NLR NRG1

et al. 2019

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Summary Most land plant genomes carry genes that encode RPW8‐NLR Resistance (R) proteins. Angiosperms carry two RPW8‐NLR subclasses: ADR1 and NRG1. ADR1s act as ‘helper’ NLRs for multiple TIR‐ and CC‐NLR R proteins in Arabidopsis. In angiosperm families, NRG1 co‐occurs with TIR‐NLR Resistance (R) genes. We tested whether NRG1 is required for signalling of multiple TIR‐NLRs. Using CRISPR mutagenesis, we obtained an nrg1a‐nrg1b double mutant in two Arabidopsis accessions, and an nrg1 mutant in Nicotiana benthamiana. These mutants are compromised in signalling of all TIR‐NLRs tested, including WRR4A, WRR4B, RPP1, RPP2, RPP4 and the pairs RRS1/RPS4, RRS1B/RPS4B, CHS1/SOC3 and CHS3/CSA1. In Arabidopsis, NRG1 is required for the hypersensitive cell death response (HR) and full oomycete resistance, but not for salicylic acid induction or bacterial resistance. By contrast, nrg1 loss of function does not compromise the CC‐NLR R proteins RPS5 and MLA. RPM1 and RPS2 (CC‐NLRs) function is slightly compromised in an nrg1 mutant. Thus, NRG1 is required for full TIR‐NLR function and contributes to the signalling of some CC‐NLRs. Some NRG1‐dependent R proteins also signal partially via the NRG1 sister clade, ADR1. We propose that some NLRs signal via NRG1 only, some via ADR1 only and some via both or neither.

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Isolation of Protein Complexes from Tobacco Leaves by a Two‐Step Tandem Affinity Purification

2022

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Protein purification is an essential method for understanding protein function, as many biochemical and structural techniques require a high concentration of isolated protein for analysis. Yet, many studies of protein complexes are hampered by our inability to express them recombinantly in model systems, generally due to poor expression or aggregation. When studying a protein complex that requires its host cellular environment for proper expression and folding, endogenous purification is typically required. Depending on the protein of interest, however, endogenous purification can be challenging because of low expression levels in the host and lack of knowledge working with a non‐model expression system, resulting in yields that are too low for subsequent analysis. Here, we describe a protocol for the purification of protein complexes endogenous to Nicotiana benthamiana directly from leaf tissue, with yields that enable structural and biochemical characterization. The protein complex is overexpressed in Nicotiana benthamiana leaves via agroinfiltration, and the protein‐packed leaves are then mechanically ground to release the complex from the cells. The protein complex is finally purified by a simple two‐step tandem affinity purification using distinct affinity tags for each complex member, to ensure purification of the assembled complex. Our method yields enough protein for various biochemical or structural studies. We have previously used this protocol to purify the complex formed by an innate immune receptor native to tobacco, ROQ1, and the Xanthomonas effector XopQ, and to solve its structure by single‐particle cryo‐electron microscopy—we use this example to illustrate the approach. This protocol may serve as a template for the purification of proteins from N. benthamiana that require the plant's cellular environment and are expressed at low levels. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expression of the protein complex in leaf tissue Basic Protocol 2: Tandem affinity purification of the ROQ1‐XopQ complex

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NRG1 functions downstream of EDS1 to regulate TIR-NLR-mediated plant immunity in Nicotiana benthamiana

Cited by 187 publications

References 56 publications

Differential regulation of TNL‐mediated immune signaling by redundant helper CNLs

Differential regulation of TNL‐mediated immune signaling by redundant helper CNLs

Diverse NLR immune receptors activate defence via the RPW8‐NLR NRG1

Isolation of Protein Complexes from Tobacco Leaves by a Two‐Step Tandem Affinity Purification

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