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Yet, in plants, the enzymes responsible for their turnover remain largely unknown. Using a non\u2010hydrolysable PP\u2010InsP analog in a pull\u2010down approach, we identified a family of Arabidopsis NUDIX\u2010type hydrolases (NUDTs) that group into two closely related subclades. Through\n                    <jats:italic>in vitro<\/jats:italic>\n                    assays, heterologous expression systems, and higher order gene\u2010edited mutants, we explored the substrate specificities and physiological roles of these hydrolases. Using a combination of strong anion exchange high\u2010performance liquid chromatography (SAX\u2010HPLC), polyacrylamide gel electrophoresis (PAGE), and capillary electrophoresis electrospray ionization mass spectrometry (CE\u2010ESI\u2010MS), we found that their PP\u2010InsP pyrophosphatase activity is enantiomer selective and Mg\n                    <jats:sup>2+<\/jats:sup>\n                    dependent. Specifically, Subclade I NUDTs preferentially hydrolyze 4\u2010InsP\n                    <jats:sub>7<\/jats:sub>\n                    , while Subclade II NUDTs target 3\u2010InsP\n                    <jats:sub>7<\/jats:sub>\n                    , with minor activity against other PP\u2010InsPs, including 5\u2010InsP\n                    <jats:sub>7<\/jats:sub>\n                    . In higher order mutants of Subclade II NUDTs, we observed defects in both P\n                    <jats:sub>i<\/jats:sub>\n                    and iron homeostasis, accompanied by increased levels of 1\/3\u2010InsP\n                    <jats:sub>7<\/jats:sub>\n                    and 5\u2010InsP\n                    <jats:sub>7<\/jats:sub>\n                    , with a markedly larger increase in 1\/3\u2010InsP\n                    <jats:sub>7<\/jats:sub>\n                    . Ectopic expression of NUDTs from both subclades induced local P\n                    <jats:sub>i<\/jats:sub>\n                    starvation responses (PSRs), while RNA\u2010seq analysis comparing wild\u2010type (WT) and Subclade II\n                    <jats:italic>nudt12\/13\/16<\/jats:italic>\n                    loss\u2010of\u2010function plants indicates additional PSR\u2010independent roles, potentially involving 1\/3\u2010InsP\n                    <jats:sub>7<\/jats:sub>\n                    in the regulation of plant defense. Consistently,\n                    <jats:italic>nudt12\/13\/16<\/jats:italic>\n                    mutants displayed enhanced resistance to\n                    <jats:italic>Pseudomonas syringae<\/jats:italic>\n                    infection, indicating a role in bacterial pathogen susceptibility. Expanding beyond Subclade II NUDTs, we demonstrated susceptibility of the 3PP\u2010position of PP\u2010InsPs to enzymatic activities unrelated to NUDTs, and found that such activities are conserved across plants and humans. Additionally, we observed that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to Subclade I NUDTs. Collectively, our findings reveal new roles for NUDTs in PP\u2010InsP signaling, plant nutrient and immune responses, and highlight a cross\u2010kingdom conservation of PP\u2010InsP\u2010metabolizing enzymes.\n                  <\/jats:p>","DOI":"10.1111\/jipb.70060","type":"journal-article","created":{"date-parts":[[2025,10,30]],"date-time":"2025-10-30T12:21:31Z","timestamp":1761826891000},"page":"3123-3151","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["NUDIX hydrolases target specific inositol pyrophosphates and regulate phosphate homeostasis and bacterial pathogen susceptibility in Arabidopsis"],"prefix":"10.1111","volume":"67","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9657-4548","authenticated-orcid":false,"given":"Robin","family":"Schneider","sequence":"first","affiliation":[{"name":"Department of Plant Nutrition Institute of Crop Science and Resource Conservation, University of Bonn Bonn 53115 Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-6750-1350","authenticated-orcid":false,"given":"Klea","family":"Lami","sequence":"additional","affiliation":[{"name":"Department of Plant Nutrition Institute of Crop Science and Resource Conservation, University of Bonn Bonn 53115 Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5794-6759","authenticated-orcid":false,"given":"Isabel","family":"Prucker","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Pharmacy and CIBSS\u2010Centre for Integrative Biological Signalling Studies Albert\u2010Ludwigs University Freiburg Freiburg 79104 Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1421-9703","authenticated-orcid":false,"given":"Sara Christina","family":"Stolze","sequence":"additional","affiliation":[{"name":"Protein Mass Spectronomy Max Planck Institute for Plant Breeding Research Cologne 50829 Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2266-6239","authenticated-orcid":false,"given":"Annett","family":"Strau\u00df","sequence":"additional","affiliation":[{"name":"Department of General Genetics Center of Plant Molecular Biology (ZMBP) University of T\u00fcbingen T\u00fcbingen 72076 Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-6783-0814","authenticated-orcid":false,"given":"Julie Marie","family":"Schmidt","sequence":"additional","affiliation":[{"name":"Department of Plant Nutrition Institute of Crop Science and Resource Conservation, University of Bonn Bonn 53115 Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2215-7901","authenticated-orcid":false,"given":"Simon 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Conservation, University of Bonn Bonn 53115 Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-1065-818X","authenticated-orcid":false,"given":"Michael","family":"Harings","sequence":"additional","affiliation":[{"name":"Department of Plant Nutrition Institute of Crop Science and Resource Conservation, University of Bonn Bonn 53115 Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8101-0558","authenticated-orcid":false,"given":"Florian M. 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