Cell Death Triggered by the YUCCA-like Bs3 Protein Coincides with Accumulation of Salicylic Acid and Pipecolic Acid But Not of Indole-3-Acetic Acid

Krönauer, Christina; Kilian, Joachim; Strauß, T.; Stahl, Mark; Lahaye, Thomas

doi:10.1104/pp.18.01576

Cited by 9 publications

(28 citation statements)

References 39 publications

(43 reference statements)

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“…This shows that the execution of an HR by the NLR protein Bs2 or the executor R protein Bs3 in pepper leaves both correlate with a local increase of H 2 O 2 levels. This observation is consistent with previous studies in Nicotiana benthamiana leaves where Agrobacterium tumefaciens mediated delivery (agroinfiltration) of a 35S promoter-driven Bs3 T-DNA caused ROS accumulation in inoculated leaf tissue after two days (Krönauer et al 2019, Fig. S1).…”

Section: Resultssupporting

confidence: 93%

“…The redox-reporter was also fused to the Bs3-derivative Bs3 S211A (mutation in NADPH binding site [G×G× S G ⟶ G×G× A G]; increased NADPH oxidase activity in vitro ; see Fig. 3) and the previously studied Bs3 derivatives Bs3 G209A (mutation in NADPH binding site [Gx G xSG ⟶ Gx A xSG]) and Bs3 G41A (mutation in FAD binding site [G× G ×SG ⟶ G× A ×SG]; Krönauer et al 2019). Analysis of these distinct fusion proteins should clarify how distinct mutations in cofactor binding sites affect NADPH oxidase activity of Bs3 in planta .…”

Section: Resultsmentioning

confidence: 99%

“…Bs3 is most closely related to YUCCA proteins, a plant-specific FMO subgroup that catalyzes the final step in tryptophan-dependent auxin (Indole-3-Acetic Acid; IAA) biosynthesis (Dai et al, 2013). The most prominent difference of Bs3 and YUCCAs is a stretch of ~70 amino acids, that is conserved across all YUCCAs but absent from Bs3 (Krönauer et al, 2019). Studies of mammalian FMOs established structure-function relationships with several motifs being conserved also in plant YUCCAs (Thodberg and Jakobsen Neilson, 2020), however, no function has been assigned to this ~70 residue stretch absent in Bs3 and ultimately its functional relevance cannot be foreseen.…”

Section: Introductionmentioning

confidence: 99%

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The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H₂O₂in vitro

Kroenauer

Lahaye

2021

Preprint

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The pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding transcription activator-like effector AvrBs3 from the bacterial pathogen Xanthomonas. Bs3 is homologous to flavin monooxygenases (FMOs), an enzyme class that has NADPH oxidase activity and can produce H2O2, a hallmark metabolite in plant immune reactions. Histochemical staining of infected pepper leaves and a translational fusion of Bs3 to the redox reporter roGFP2 both indicated that the Bs3-dependent HR induces a local increase in H2O2 levels in planta. Moreover, our in vitro studies with recombinant Bs3 protein confirmed its NADPH oxidase activity. To test if the NADPH oxidation of Bs3 induces HR, we adapted previous studies which have uncovered mutations in fungal FMOs that result in higher NADPH oxidase activity. We replicated one of these mutations and demonstrated that the generated recombinant Bs3S211A protein has twofold higher NADPH oxidase activity than wildtype Bs3 in vitro. Translational fusions to roGFP2 showed that Bs3S211A also had increased NADPH oxidase activity in planta. Interestingly, while the mutant derivative Bs3S211A had an increase in NADPH oxidase capacity, it did not trigger HR in planta. Ultimately, this reveals that Bs3 produces H2O2in planta, but that the H2O2 produced by Bs3 on its own is not sufficient to trigger HR. We also demonstrated that expression of Bs3 not only triggered HR in plants, but also inhibited proliferation of yeast, which lends this model system to be utilized for the genetic dissection of Bs3 function in future studies.

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H₂O₂in vitro

Kroenauer

Lahaye

2021

Preprint

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“…Site-directed mutagenesis of this residue disrupted the TR functionality of YUC6; however, the ability to catalyze the conversion of IPA to IAA was still maintained. This striking dual role of YUC6 illuminates many possibilities to investigate additional functionalities of other YUCCA and YUCCA-like proteins [79], but also illustrates that caution needs to be taken when interpreting physiological output in knockout and over-expressing YUCCA lines. Despite the overall progress in YUCCA functionality within Arabidopsis and other model plant species, surprisingly little is known about the biochemical mechanisms of these proteins, as well as broader catalytic properties such as substrate specificity.…”

Section: Yuccasmentioning

confidence: 95%

The “Green” FMOs: Diversity, Functionality and Application of Plant Flavoproteins

Thodberg

Neilson

2020

Catalysts

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Flavin-dependent monooxygenases (FMOs) are ancient enzymes present in all kingdoms of life. FMOs typically catalyze the incorporation of an oxygen atom from molecular oxygen into small molecules. To date, the majority of functional characterization studies have been performed on mammalian, fungal and bacterial FMOs, showing that they play fundamental roles in drug and xenobiotic metabolism. By contrast, our understanding of FMOs across the plant kingdom is very limited, despite plants possessing far greater FMO diversity compared to both bacteria and other multicellular organisms. Here, we review the progress of plant FMO research, with a focus on FMO diversity and functionality. Significantly, of the FMOs characterized to date, they all perform oxygenation reactions that are crucial steps within hormone metabolism, pathogen resistance, signaling and chemical defense. This demonstrates the fundamental role FMOs have within plant metabolism, and presents significant opportunities for future research pursuits and downstream applications.

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“…With the exception of rice Xa10 and Xa23, which share 50% identity, the predicted executor R proteins show no homology to each other. Recent studies of the executor R protein Bs3 from pepper revealed that the Bs3-triggered immune reaction involves accumulation of salicylic acid (SA), a plant defence hormone that is involved in NLR-and PRR-triggered immune pathways (15). These findings possibly suggest that NLR-, PRR-and executor-type R proteins use, at least in part, common signalling elements to trigger plant defence.…”

Section: Introductionmentioning

confidence: 99%

A shortcut in forward genetics: concurrent discovery of mutant phenotype and causal mutation in Arabidopsis M2 families via MAD-mapping

Holmes

Morbitzer

Wunderlich

et al. 2020

Preprint

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ABSTRACTForward genetics is a powerful tool to establish phenotype-genotype correlations in virtually all areas of plant biology and has been particularly successful in the model plant Arabidopsis. This approach typically starts with a phenotype in an M2 mutant, followed by identifying a causal DNA change in F2 populations resulting from a cross between the mutant and a wildtype individual. Ultimately, two additional generations are needed to pinpoint causal DNA changes upon mutant identification. We postulated that genome-wide allele frequency distributions within the mutants of M2 families facilitate discrimination of causal versus non-causal mutations, essentially eliminating the need for F2 populations. In a proof-of-principle experiment, we aimed to identify signalling components employed by the executor-type resistance (R) protein, Bs4C, from pepper (Capsicum pubescens). In a native setting, Bs4C is transcriptionally activated by and mediates recognition of the transcription activator-like effector AvrBs4 from the bacterial pathogen Xanthomonas. Arabidopsis containing an estradiol-inducible Bs4C transgene was used in a conditionally lethal screen to identify second-site suppressor mutations. Whole genome sequencing was used for M2 mutant allele-frequency distribution (MAD) mapping in three independent M2 families. MAD-mapping uncovered that all three families harboured mutations in XRN4, a novel component of executor R protein pathways. Our work demonstrates that causal mutations observed in forward genetic screens can be identified immediately in M2 families instead of derived F2 families. Notably, the timesaving concept of MAD mapping should be applicable to most crop species and will advance the appeal of forward genetics beyond applications in fundamental research.SIGNIFICANCEForward genetics has uncovered numerous genes that govern plant immune reactions. This procedure relies on mutant plants with modified immune reactions followed by identification of causal DNA changes in derived F2 progeny. We developed a novel forward genetics concept where causal DNA changes are identified in the initial M2 mutants, making time consuming establishment of F2 populations obsolete. To confirm the feasibility of the concept, we mutagenized transgenic Arabidopsis seeds containing the cell death executing resistance gene Bs4C from pepper. Whole-genome sequencing of identified mutant families that lack a Bs4C-dependent cell death revealed the XRN4 gene as a novel component of Bs4C-dependent cell death. This confirms our hypothesis that causal mutations can be identified directly within phenotypically selected mutant families.

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Cell Death Triggered by the YUCCA-like Bs3 Protein Coincides with Accumulation of Salicylic Acid and Pipecolic Acid But Not of Indole-3-Acetic Acid

Cited by 9 publications

References 39 publications

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H₂O₂in vitro

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H₂O₂in vitro

The “Green” FMOs: Diversity, Functionality and Application of Plant Flavoproteins

A shortcut in forward genetics: concurrent discovery of mutant phenotype and causal mutation in Arabidopsis M2 families via MAD-mapping

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Cell Death Triggered by the YUCCA-like Bs3 Protein Coincides with Accumulation of Salicylic Acid and Pipecolic Acid But Not of Indole-3-Acetic Acid

Cited by 9 publications

References 39 publications

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2in vitro

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2in vitro

The “Green” FMOs: Diversity, Functionality and Application of Plant Flavoproteins

A shortcut in forward genetics: concurrent discovery of mutant phenotype and causal mutation in Arabidopsis M2 families via MAD-mapping

Contact Info

Product

Resources

About

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H₂O₂in vitro

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H₂O₂in vitro