The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues

Molekulare und funktionelle Charakterisierung des Arabidopsis thaliana Response Regulators ARR18

The two-component signaling system evolved as an important sensing and responding mechanism in Arabidopsis thaliana, which consists of hybrid histidine kinases (AHKs), histidine phosphotransfer proteins (AHPs) and response regulators (ARRs) biochemically linked by a histidine to aspartate phosphorelay. In our work, several functional and molecular biological approaches were performed to characterize and understand the function of the B-type response regulator ARR18. In our study, ARR18 has been found to be an active transcription factor whose activity and homodimerization mainly depend on its phosphorylation state. Further investigations revealed that ARR18 is also able to form heteromers with bZIP63 and negatively influences the transcriptional activity of bZIP63 in a process that again depends on the ARR18 phosphorylation state. Moreover, extensive studies showed that ARR18 and bZIP63 function as antagonistic regulators in osmotic stress signaling in Arabidopsis thaliana. The results of our study reveal the importance of the phosphorylation state of the ARRs in controlling each signaling mechanism and the importance of non-TCS related proteins which, by direct interaction with TCS elements, add a new degree of diversity and complexity to the TCS signaling network.Das Zweikomponentensystem hat sich zu einem wichtigen Signal-Antwort-Mechanismus in Arabidopsis thaliana entwickelt und besteht aus Hybrid-Histidinkinasen (AHKs), Histidin Phosphotransferproteinen (AHPs) und Responseregulatoren (ARRs), welche biochemisch über einen Histidin zu Aspartat Phosphorelay miteinander in Verbindung stehen. In der vorliegenden Arbeit wurde die funktion des Response-reulators ARR18 durch verschiedene funktionale und molekularbiologische Ansätze näher characterisiert. Es konnte gezeigt werden, dass ARR18 ein aktiver Transkriptionsfaktor ist dessen Aktivität und Homodimerisierung hauptsächlich von seinem Phosphorylierungszustand abhängen. Weitere Untersuchungen zeigten, dass ARR18 auch Heterodimere mit bZIP63 bilden kann und dessen Transkriptionsaktivität negativ beeinflusst, was ebenfalls vom Phosphorylierungszustand von ARR18 abhängt. Des Weiteren wurde durch umfassende Untersuchungen gezeigt, dass ARR18 und bZIP63 antagonistische Regulatoren der osmotischen Stressantwort in Arabidopsis thaliana darstellen. Die Ergebnisse unserer Untersuchungen zeigen die Bedeutung des Phosphorylierungszustandes der ARR18 bei der Kontrolle jedes Signalmechanismus sowie die Bedeutung von Proteinen, die nicht zum Zweikomponentensystem gehören jedoch durch direkte Interaktion mit dessen Elementen diesem ein neues Ausmaß an Diversität und Komplexität verleihen

Plant lipid bodies traffic on actin to plasmodesmata motorized by myosin XIs

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ARR22 overexpression can suppress plant Two-Component Regulatory Systems

In plants, several developmental processes are co-coordinated by cytokinins via phosphorylation dependent processes of the Two-Component System (TCS). An outstanding challenge is to track phosphorelay flow from cytokinin perception to its molecular outputs, of which gene activation plays a major role. To address this issue, a kinetic-based reporter system was expounded to track TCS phosphorelay activity in vivo that can distinguish between basal and cytokinin dependent effects of overexpressed TCS members. The TCS phosphorelay can be positively activated by cytokinin and inhibited by pharmaceuticals or naturally interfering components. In this case we took advantage of the phosphohistidinephosphatase Arabidopsis Response Regulator (ARR) 22 and investigated its phosphocompetition with other TCS members in regulating promoters of ARR5 and WUS in Arabidopsis thaliana cell culture protoplasts. In congruency with the proposed function of ARR22, overexpression of ARR22 blocked the activation of all B-type ARRs in this study in a TCS dependent manner. Furthermore, this effect could not be mimicked by A-type response regulator overexpression or compensated by AHP overexpression. Compared to other reporter assays, ours mimicked effects previously observed only in transgenic plants for all of the TCS proteins studied, suggesting that it is possible to expose phosphocompetition. Thus, our approach can be used to investigate gene signaling networks involving the TCS by leveraging ARR22 as a TCS inhibitor along with B-type ARR overexpression.publishedVersio

Plant lipid bodies traffic on actin to plasmodesmata motorized by myosin XIs

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Lipid body dynamics in shoot meristems: production, enlargement, and putative organellar interactions and plasmodesmal targeting

Post-embryonic cells contain minute lipid bodies (LBs) that are transient, mobile, engage in organellar interactions, and target plasmodesmata (PD). While LBs can deliver γ‐clade 1,3‐β‐glucanases to PD, the nature of other cargo is elusive. To gain insight into the poorly understood role of LBs in meristems, we investigated their dynamics by microscopy, gene expression analyzes and proteomics. In developing buds, meristems accumulated LBs, upregulated several LB-specific OLEOSIN genes, and produced OLEOSINs. During bud maturation, the major gene OLE6 was strongly downregulated, OLEOSINs disappeared from bud extracts, whereas lipid biosynthesis genes were upregulated, and LBs enlarged. Proteomic analyses of the LB fraction of dormant buds confirmed that OLEOSINs were no longer present. Instead, we identified the LB-associated proteins CALEOSIN (CLO1), Oil Body Lipase 1 (OBL1), Lipid Droplet Interacting Protein (LDIP), Lipid Droplet Associated Protein1a/b (LDAP1a/b) and LDAP3a/b, and crucial components of the OLEOSIN-deubiquitinating and degradation machinery, including PUX10 and CDC48A. All RFP-tagged LDAPs localized to LBs when transiently expressed in Nicotiana benthamiana. Together with gene expression analyzes this suggests that during bud maturation OLEOSINs were replaced by LDIP/LDAPs on enlarging LBs. The LB fraction contained the meristem-related actin7 (ACT7), ‘myosin XI tail‐binding’ RAB GTPase C2A, a LB/PD‐associated γ‐clade 1,3‐β‐glucanase, various organelle‐ and/or PD‐localized proteins. The results are congruent with a model in which LBs, motorized by myosin XI-k/1/2, traffic on F-actin, transiently interact with other organelles and deliver a diverse cargo to PD

The plant cell wall integrity maintenance and immune signaling systems cooperate to control stress responses in Arabidopsis thaliana

Cell walls surround all plant cells, and their composition and structure are modified in a tightly controlled, adaptive manner to meet sometimes opposing functional requirements during growth and development. The plant cell wall integrity (CWI) maintenance mechanism controls these functional modifications, as well as responses to cell wall damage (CWD). We investigated how the CWI system mediates responses to CWD in Arabidopsis thaliana. CWD induced by cell wall–degrading enzymes or an inhibitor of cellulose biosynthesis elicited similar, turgor-sensitive stress responses. Phenotypic clustering with 27 genotypes identified a core group of receptor-like kinases (RLKs) and ion channels required for the activation of CWD responses. A genetic analysis showed that the RLK FEI2 and the plasma membrane–localized mechanosensitive Ca2+ channel MCA1 functioned downstream of the RLK THE1 in CWD perception. In contrast, pattern-triggered immunity (PTI) signaling components, including the receptors for plant elicitor peptides (AtPeps) PEPR1 and PEPR2, repressed responses to CWD. CWD induced the expression of PROPEP1 and PROPEP3, which encode the precursors of AtPep1 and AtPep3, and the release of PROPEP3 into the growth medium. Application of AtPep1 and AtPep3 repressed CWD-induced phytohormone accumulation in a concentration-dependent manner. These results suggest that AtPep-mediated signaling suppresses CWD-induced defense responses controlled by the CWI mechanism. This suppression was alleviated when PTI signaling downstream of PEPR1 and PEPR2 was impaired. Defense responses controlled by the CWI maintenance mechanism might thus compensate to some extent for the loss of PTI signaling elements