The Tomato NBARC-LRR Protein Prf Interacts with Pto Kinase in Vivo to Regulate Specific Plant Immunity

Abstract Protein ubiquitylation profoundly expands proteome functionality and diversifies cellular signaling processes, with recent studies providing ample evidence for its importance to plant immunity. To gain a proteome-wide appreciation of ubiquitylome dynamics during immune recognition, we employed a two-step affinity enrichment protocol based on a 6His-tagged ubiquitin (Ub) variant coupled with high sensitivity mass spectrometry to identify Arabidopsis proteins rapidly ubiquitylated upon plant perception of the microbe-associated molecular pattern (MAMP) peptide flg22. The catalog from 2-week-old seedlings treated for 30 minutes with flg22 contained 690 conjugates, 64 Ub footprints, and all seven types of Ub linkages, and included previously uncharacterized conjugates of immune components. In vivo ubiquitylation assays confirmed modification of several candidates upon immune elicitation, and revealed distinct modification patterns and dynamics for key immune components, including poly- and monoubiquitylation, as well as induced or reduced levels of ubiquitylation. Gene ontology and network analyses of the collection also uncovered rapid modification of the Ub-proteasome system itself, suggesting a critical auto-regulatory loop necessary for an effective MAMP-triggered immune response and subsequent disease resistance. Included targets were UBIQUITIN-CONJUGATING ENZYME 13 (UBC13) and proteasome component REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b), whose subsequent biochemical and genetic analyses implied negative roles in immune elicitation. Collectively, our proteomic analyses further strengthened the connection between ubiquitylation and flg22-based immune signaling, identified components and pathways regulating plant immunity, and increased the database of ubiquitylated substrates in plants.

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Disruption of PAMP-Induced MAP Kinase Cascade by a Pseudomonas syringae Effector Activates Plant Immunity Mediated by the NB-LRR Protein SUMM2

Cell Host & Microbe ◽

10.1016/j.chom.2012.01.015 ◽

2012 ◽

Vol 11 (3) ◽

pp. 253-263 ◽

Cited By ~ 171

Author(s):

Zhibin Zhang ◽

Yaling Wu ◽

Minghui Gao ◽

Jie Zhang ◽

Qing Kong ◽

...

Keyword(s):

Map Kinase ◽

Pseudomonas Syringae ◽

Plant Immunity ◽

Map Kinase Cascade ◽

Kinase Cascade ◽

Lrr Protein

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A vector system for fast-forward studies of the HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome in the model plant Nicotiana benthamiana

Plant Physiology ◽

10.1093/plphys/kiab471 ◽

2021 ◽

Author(s):

Adeline Harant ◽

Hsuan Pai ◽

Toshiyuki Sakai ◽

Sophien Kamoun ◽

Hiroaki Adachi

Keyword(s):

Nicotiana Benthamiana ◽

Cell Biology ◽

Plant Immunity ◽

Transient Gene Expression ◽

Coiled Coil ◽

Experimental System ◽

Vector System ◽

In Planta ◽

Functional Studies

Abstract Nicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis thaliana. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of nucleotide-binding leucine-rich repeat (NLR) plant immune receptors has greatly advanced following the discovery of the Arabidopsis HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome. Here, we describe a vector system of 72 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs (CC-NLRs) for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. Effector-activated and autoactive NbZAR1 trigger the cell death response in N. benthamiana and this activity is dependent on a functional N-terminal α1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana, thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source pZA plasmid collection forms an additional experimental system to Arabidopsis for in planta resistosome studies.

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Extracellular pyridine nucleotides trigger plant systemic immunity through a lectin receptor kinase/BAK1 complex

Nature Communications ◽

10.1038/s41467-019-12781-7 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 12

Author(s):

Chenggang Wang ◽

Xiaoen Huang ◽

Qi Li ◽

Yanping Zhang ◽

Jian-Liang Li ◽

...

Keyword(s):

Nicotinamide Adenine Dinucleotide ◽

Systemic Acquired Resistance ◽

Plant Immunity ◽

Acquired Resistance ◽

Receptor Complex ◽

Pyridine Nucleotides ◽

Receptor Kinase ◽

Systemic Immunity ◽

Lectin Receptor

Abstract Systemic acquired resistance (SAR) is a long-lasting broad-spectrum plant immunity induced by mobile signals produced in the local leaves where the initial infection occurs. Although multiple structurally unrelated signals have been proposed, the mechanisms responsible for perception of these signals in the systemic leaves are unknown. Here, we show that exogenously applied nicotinamide adenine dinucleotide (NAD+) moves systemically and induces systemic immunity. We demonstrate that the lectin receptor kinase (LecRK), LecRK-VI.2, is a potential receptor for extracellular NAD+ (eNAD+) and NAD+ phosphate (eNADP+) and plays a central role in biological induction of SAR. LecRK-VI.2 constitutively associates with BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) in vivo. Furthermore, BAK1 and its homolog BAK1-LIKE1 are required for eNAD(P)+ signaling and SAR, and the kinase activities of LecR-VI.2 and BAK1 are indispensable to their function in SAR. Our results indicate that eNAD+ is a putative mobile signal, which triggers SAR through its receptor complex LecRK-VI.2/BAK1 in Arabidopsis thaliana.

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Cysteine protease RD21A regulated by E3 ligase SINAT4 is required for drought-induced resistance to Pseudomonas syringae in Arabidopsis

Journal of Experimental Botany ◽

10.1093/jxb/eraa255 ◽

2020 ◽

Vol 71 (18) ◽

pp. 5562-5576

Author(s):

Yi Liu ◽

Kunru Wang ◽

Qiang Cheng ◽

Danyu Kong ◽

Xunzhong Zhang ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Cysteine Protease ◽

Plant Immunity ◽

Stomatal Closure ◽

Bacterial Pathogen ◽

E3 Ligase ◽

Biotic Stresses ◽

Ubiquitin E3 Ligase ◽

Induced Immunity

Abstract Plants can be simultaneously exposed to multiple stresses. The interplay of abiotic and biotic stresses may result in synergistic or antagonistic effects on plant development and health. Temporary drought stress can stimulate plant immunity; however, the molecular mechanism of drought-induced immunity is largely unknown. In this study, we demonstrate that cysteine protease RD21A is required for drought-induced immunity. Temporarily drought-treated wild-type Arabidopsis plants became more sensitive to the bacterial pathogen-associated molecular pattern flg22, triggering stomatal closure, which resulted in increased resistance to Pseudomonas syringae pv. tomato DC3000 (Pst-DC3000). Knocking out rd21a inhibited flg22-triggered stomatal closure and compromised the drought-induced immunity. Ubiquitin E3 ligase SINAT4 interacted with RD21A and promoted its degradation in vivo. The overexpression of SINAT4 also consistently compromised the drought-induced immunity to Pst-DC3000. A bacterial type III effector, AvrRxo1, interacted with both SINAT4 and RD21A, enhancing SINAT4 activity and promoting the degradation of RD21A in vivo. Therefore, RD21A could be a positive regulator of drought-induced immunity, which could be targeted by pathogen virulence effectors during pathogenesis.

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Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1504154112 ◽

2015 ◽

Vol 112 (17) ◽

pp. 5533-5538 ◽

Cited By ~ 81

Author(s):

Manuel Benedetti ◽

Daniela Pontiggia ◽

Sara Raggi ◽

Zhenyu Cheng ◽

Flavio Scaloni ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Plant Immunity ◽

Inducible Promoter ◽

Spectrometric Analysis ◽

In Planta ◽

Gene Encoding ◽

Polygalacturonase Inhibiting Protein ◽

Molecular Patterns ◽

Damage Associated Molecular Patterns

Oligogalacturonides (OGs) are fragments of pectin that activate plant innate immunity by functioning as damage-associated molecular patterns (DAMPs). We set out to test the hypothesis that OGs are generated in planta by partial inhibition of pathogen-encoded polygalacturonases (PGs). A gene encoding a fungal PG was fused with a gene encoding a plant polygalacturonase-inhibiting protein (PGIP) and expressed in transgenic Arabidopsis plants. We show that expression of the PGIP–PG chimera results in the in vivo production of OGs that can be detected by mass spectrometric analysis. Transgenic plants expressing the chimera under control of a pathogen-inducible promoter are more resistant to the phytopathogens Botrytis cinerea, Pectobacterium carotovorum, and Pseudomonas syringae. These data provide strong evidence for the hypothesis that OGs released in vivo act as a DAMP signal to trigger plant immunity and suggest that controlled release of these molecules upon infection may be a valuable tool to protect plants against infectious diseases. On the other hand, elevated levels of expression of the chimera cause the accumulation of salicylic acid, reduced growth, and eventually lead to plant death, consistent with the current notion that trade-off occurs between growth and defense.

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A putative nuclear copper chaperone promotes plant immunity in Arabidopsis

Journal of Experimental Botany ◽

10.1093/jxb/eraa401 ◽

2020 ◽

Vol 71 (20) ◽

pp. 6684-6696 ◽

Cited By ~ 1

Author(s):

Long-Xiang Chai ◽

Kai Dong ◽

Song-Yu Liu ◽

Zhen Zhang ◽

Xiao-Peng Zhang ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Plant Immunity ◽

Copper Chaperone ◽

Copper Binding ◽

Nuclear Speckles ◽

Defense Gene Expression ◽

Copper Chaperones ◽

Plant Nucleus

Abstract Copper is essential for many metabolic processes but must be sequestrated by copper chaperones. It is well known that plant copper chaperones regulate various physiological processes. However, the functions of copper chaperones in the plant nucleus remain largely unknown. Here, we identified a putative copper chaperone induced by pathogens (CCP) in Arabidopsis thaliana. CCP harbors a classical MXCXXC copper-binding site (CBS) at its N-terminus and a nuclear localization signal (NLS) at its C-terminus. CCP mainly formed nuclear speckles in the plant nucleus, which requires the NLS and CBS domains. Overexpression of CCP induced PR1 expression and enhanced resistance against Pseudomonas syringae pv. tomato DC3000 compared with Col-0 plants. Conversely, two CRISPR/Cas9-mediated ccp mutants were impaired in plant immunity. Further biochemical analyses revealed that CCP interacted with the transcription factor TGA2 in vivo and in vitro. Moreover, CCP recruits TGA2 to the PR1 promoter sequences in vivo, which induces defense gene expression and plant immunity. Collectively, our results have identified a putative nuclear copper chaperone required for plant immunity and provided evidence for a potential function of copper in the salicylic pathway.

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Phosphatase PP2C6 negatively regulates phosphorylation status of Pti1b kinase, a regulator of flagellin-triggered immunity in tomato

10.1101/613612 ◽

2019 ◽

Author(s):

Fabian Giska ◽

Gregory B. Martin

Keyword(s):

Immune Responses ◽

Plant Immunity ◽

Active Form ◽

Control Plant ◽

Negative Regulator ◽

Response To Treatment ◽

Positive Regulators ◽

Molecular Patterns

AbstractPlant immune responses, including the production of reactive oxygen species (ROS), are triggered when pattern recognition receptors (PRR) become activated upon detection of microbe-associated molecular patterns (MAMPs). Receptor-like cytoplasmic kinases are key components of PRR-dependent signaling pathways. In tomato two such kinases, Pti1a and Pti1b, are important positive regulators of the plant immune response. However, it is unknown how these kinases control plant immunity at the molecular level, and how their activity is regulated. To investigate these issues, we used mass spectrometry to search for interactors of Pti1b in Nicotiana benthamiana leaves and identified a protein phosphatase, PP2C6. An in vitro pull-down assay and in vivo split luciferase complementation assay verified this interaction. Pti1b was found to autophosphorylate on threonine-233 and this phosphorylation was abolished in the presence of PP2C6. An arginine-to-cysteine substitution at position 240 in the Arabidopsis MARIS kinase was previously reported to convert it into a constitutive-active form. The analogous substitution in Pti1b made it resistant to PP2C6 phosphatase activity, although it still interacted with PP2C6. Treatment of N. benthamiana leaves with the MAMP flg22 induced threonine phosphorylation of Pti1b. Expression of PP2C6, but not a phosphatase-inactive variant of this protein, in N. benthamiana leaves greatly reduced ROS production in response to treatment with MAMPs flg22 or csp22. The results indicate that PP2C6 acts as a negative regulator by dephosphorylating the Pti1b kinase, thereby interfering with its ability to activate plant immune responses.

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A Phytophthora sojae CRN effector mediates phosphorylation and degradation of plant aquaporin proteins to suppress host immune signaling

PLoS Pathogens ◽

10.1371/journal.ppat.1009388 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009388

Author(s):

Gan Ai ◽

Qingyue Xia ◽

Tianqiao Song ◽

Tianli Li ◽

Hai Zhu ◽

...

Keyword(s):

Higher Plants ◽

Plant Immunity ◽

Phytophthora Capsici ◽

Kinase Domain ◽

Phytophthora Sojae ◽

Effector Proteins ◽

Ros Accumulation ◽

Oomycete Pathogen ◽

Cellular Defenses

Phytophthora genomes encode a myriad of Crinkler (CRN) effectors, some of which contain putative kinase domains. Little is known about the host targets of these kinase-domain-containing CRNs and their infection-promoting mechanisms. Here, we report the host target and functional mechanism of a conserved kinase CRN effector named CRN78 in a notorious oomycete pathogen, Phytophthora sojae. CRN78 promotes Phytophthora capsici infection in Nicotiana benthamiana and enhances P. sojae virulence on the host plant Glycine max by inhibiting plant H2O2 accumulation and immunity-related gene expression. Further investigation reveals that CRN78 interacts with PIP2-family aquaporin proteins including NbPIP2;2 from N. benthamiana and GmPIP2-13 from soybean on the plant plasma membrane, and membrane localization is necessary for virulence of CRN78. Next, CRN78 promotes phosphorylation of NbPIP2;2 or GmPIP2-13 using its kinase domain in vivo, leading to their subsequent protein degradation in a 26S-dependent pathway. Our data also demonstrates that NbPIP2;2 acts as a H2O2 transporter to positively regulate plant immunity and reactive oxygen species (ROS) accumulation. Phylogenetic analysis suggests that the phosphorylation sites of PIP2 proteins and the kinase domains of CRN78 homologs are highly conserved among higher plants and oomycete pathogens, respectively. Therefore, this study elucidates a conserved and novel pathway used by effector proteins to inhibit host cellular defenses by targeting and hijacking phosphorylation of plant aquaporin proteins.

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