Elicitin-responsive lectin-like receptor kinase genes in BY-2 cells

The inhibition of elicitor-induced plant defense responses by the protein kinase inhibitors K252a and staurosporine indicates that defense responses require protein phosphorylation. We isolated a cDNA clone encoding Nicotiana tabacum lectin-like receptor protein kinase 1 ( NtlecRK1), an elicitor-responsive gene; in tobacco bright yellow ( BY-2) cells by a differential display method. NtlecRK forms a gene family with at least three members in tobacco. All three NtlecRK genes potentially encode the N-terminal legume lectin domain, transmembrane domain and C-terminal Ser/Thr-type protein kinase domain. Green fluorescent protein ( GFP) fusion showed that the NtlecRK1 protein was located on the plasma membrane. In addition, NtlecRK1 and 3 were responsive to INF1 elicitin and the bacterial elicitor harpin. These results indicate that NtlecRKs are membrane-located protein kinases that are induced during defense responses in BY-2 cells.</p

BEX1/ARF1A1C is required for BFA-sensitive recycling of PIN auxin transporters and auxin-mediated development in arabidopsis

Correct positioning of membrane proteins is an essential process in eukaryotic organisms. The plant hormone auxin is distributed through intercellular transport and triggers various cellular responses. Auxin transporters of the PIN-FORMED (PIN) family localize asymmetrically at the plasma membrane (PM) and mediate the directional transport of auxin between cells. A fungal toxin, brefeldin A (BFA), inhibits a subset of guanine nucleotide exchange factors for ADP-ribosylation factor small GTPases (ARF GEFs) including GNOM, which plays a major role in localization of PIN1 predominantly to the basal side of the PM. The Arabidopsis genome encodes 19 ARF-related putative GTPases. However, ARF components involved in PIN1 localization have been genetically poorly defined. Using a fluorescence imaging-based forward genetic approach, we identified an Arabidopsis mutant, bfa-visualized exocytic trafficking defective1 (bex1), in which PM localization of PIN1-green fluorescent protein (GFP) as well as development is hypersensitive to BFA. We found that in bex1 a member of the ARF1 gene family, ARF1A1C, was mutated. ARF1A1C localizes to the trans-Golgi network/early endosome and Golgi apparatus, acts synergistically to BEN1/MIN7 ARF GEF and is important for PIN recycling to the PM. Consistent with the developmental importance of PIN proteins, functional interference with ARF1 resulted in an impaired auxin response gradient and various developmental defects including embryonic patterning defects and growth arrest. Our results show that ARF1A1C is essential for recycling of PIN auxin transporters and for various auxin-dependent developmental processes

HINKEL kinesin, ANP MAPKKKs and MKK6/ANQ MAPKK, which phosphorylates and activates MPK4 MAPK, constitute a pathway that is required for cytokinesis in Arabidopsis thaliana

Cytokinesis is regulated to ensure the precise partitioning of cytoplasm and duplicated chromosomes to daughter cells. The NACK-PQR pathway, which includes NACK1 kinesin-like protein (KLP) and a mitogen-activated protein kinase (MAPK) cascade, plays a key role in cytokinesis in tobacco cells. Although HINKEL/AtNACK1 (HIK) KLP, ANP MAP kinase kinase kinases (MAPKKKs) and MKK6/ ANQ MAP kinase kinase (MAPKK) have been identified independently as regulators of cytokinesis in Arabidopsis thaliana, the involvement of HIK, ANPs and MKK6/ANQ in a regulatory cascade remains to be demonstrated. Here we provide details of the protein kinase pathway that controls cytokinesis in A. thaliana. Analysis of the subcellular distribution of six MAPKKs of A. thaliana that had been fused to green fluorescent protein revealed that only MKK6/ANQ protein was concentrated at the equatorial plane of the phragmoplast, at the site of localization of HIK. Expression of MKK6/ANQ in yeast cells replaced the growth-control function of the MAPKK encoded by yeast PBS2, provided that both ANP1 MAPKKK and HIK [or TETRASPORE/AtNACK2 (TES)] KLP were coexpressed, suggesting that ANP1 activates MKK6/ANQ in the presence of HIK (or TES). Coexpression of HIK and ANP3 (another member of the ANP MAPKKK family) weakly activated MKK6/ANQ but that of TES and ANP3 did not. MKK6/ANQ phosphorylated MPK4 MAPK in vitro to activate the latter kinase. Thus cytokinesis in A. thaliana is controlled by a pathway that consists of ANP MAPKKKs that can be activated by HIK and MKK6/ANQ MAPKK, with MPK4 MAPK being a probable target of MKK6/ANQ

Suppression of Cdc27B expression induces plant defence responses

Non-host resistance is the most general form of disease resistance in plants because it is effective against most phytopathogens. The importance of hypersensitive responses (HRs) in non-host resistance of Nicotiana species to the oomycete Phytophthora is clear. INF1 elicitin, an elicitor obtained from the late-blight pathogen Phytophthora infestans, is sufficient to induce a typical HR in Nicotiana species. The molecular mechanisms that underlie the non-host resistance component of plant defence responses have been investigated using differential-display polymerase chain reaction (PCR) in a model HR system between INF1 elicitin and tobacco BY-2 cells. Differential-display PCR has revealed that Cdc27B is down-regulated in tobacco BY- 2 cells after treatment with INF1 elicitin. Cdc27B is one of 13 essential components of the anaphase- promoting complex or cyclosome ( APC/ C)-type E3 ubiquitin ligase complex in yeast. This APC/C-type E3 ubiquitin ligase complex regulates G2-to-M phase transition of the cell cycle by proteolytic degradation. In this study, we investigated the roles of this gene, NbCdc27B, in plant defence responses using virus-induced gene silencing. Suppression of NbCdc27B in Nicotiana benthamiana plants induced defence responses and a gain of resistance to Colletotrichum lagenarium fungus. Elicitin-induced hypersensitive cell death (HCD) was inhibited mildly in plants silenced with tobacco rattle virus:: Cdc27B. Cdc27B could manage the signalling pathways of plant defence responses as a negative regulator without HCD.</p

Amino acid substitutions in CPC-LIKE MYB reveal residues important for protein stability in Arabidopsis roots.

TRYPTICHON (TRY) and ENHANCER OF TRY AND CPC2 (ETC2) encode R3-type MYB transcription factors that are involved in epidermal cell differentiation in Arabidopsis thaliana. TRY and ETC2 belong to the CPC-like MYB gene family, which includes seven homolog genes. Previously, we showed that among the CPC family members, TRY and ETC2 are characterized by rapid proteolysis compared with that of other members, and we demonstrated that this proteolysis is mediated by the proteasome-dependent pathway. In this study, we compared the functions of the wild-type TRY and ETC2 proteins and their amino acid-substituted versions. Our results showed that the substitution of amino acids in the C-terminal of TRY and ETC2 conferred them the ability to induce root hair formation. Furthermore, we confirmed that these mutations enhanced the stability of the TRY and ETC2 proteins. These results revealed that the amino acids, which are important for the functions of TRY and ETC2, mediate morphological pattern formation and can be useful in understanding the pathway determining the fate of root hair cells

Arabidopsis MAP65-4 plays a role in phragmoplast microtubule organization and marks the cortical cell division site.

The evolutionarily conserved MAP65 family proteins bundle anti-parallel microtubules (MTs). In Arabidopsis thaliana, mutations in the MAP65-3 gene lead to serious defects in MT organization in the phragmoplast and cause failures in cytokinesis. However, the functions of other ArabidopsisMAP65 isoforms are largely unknown. MAP65 functions were analyzed based on genetic interactions among different map65 mutations. Live-cell imaging and immunolocalization experiments revealed dynamic activities of two closely related MAP65 proteins in dividing cells. The map65-4 mutation caused synthetic lethality with map65-3 although map65-4 alone did not cause a noticeable phenotype. Furthermore, the introduction of an extra copy of the MAP65-4 gene significantly suppressed defects in cytokinesis and seedling growth caused by map65-3 because of restoring MT engagement in the spindle midzone. During mitosis, MAP65-4 first appeared at the preprophase band and persisted at the cortical division site afterwards. It was also concentrated on MTs in the spindle midzone and the phragmoplast. In the absence of MAP65-3, MAP65-4 exhibited greatly enhanced localization in the midzone of developing phragmoplast. Therefore, we have uncovered redundant but differential contributions of MAP65-3 and MAP65-4 to engaging and bundling anti-parallel MTs in the phragmoplast and disclosed a novel action of MAP65-4 at the cortical cell division site

The MAP Kinase MPK4 Is Required for Cytokinesis in Arabidopsis thaliana

International audienceCytokinesis in plants is achieved by the formation of the cell plate. A pathway that includes mitogen-activated protein ( MAP) kinase kinase kinase and MAP kinase kinase (MAPKK) plays a key role in the control of plant cytokinesis. We show here that a MAP kinase, MPK4, is required for the formation of the cell plate in Arabidopsis thaliana. Single mutations in MPK4 caused dwarfism and characteristic defects in cytokinesis, such as immature cell plates, which became much more prominent upon introduction of a mutation in MKK6/ANQ, the MAPKK for cytokinesis, into mpk4. MKK6/ANQ strongly activated MPK4 in protoplasts, and kinase activity of MPK4 was detected in wild-type tissues that contained dividing cells but not in mkk6/anq mutants. Fluorescent protein-fused MPK4 localized to the expanding cell plates in cells of root tips. Expansion of the cell plates in mpk4 root tips appeared to be retarded. The level of MPK11 transcripts was markedly elevated in mpk4 plants, and defects in the mpk4 mpk11 double mutant with respect to growth and cytokinesis were more severe than in the corresponding single mutants. These results indicate that MPK4 is the downstream target of MKK6/ANQ in the regulation of cytokinesis in Arabidopsis and that MPK11 is also involved in cytokinesis

Arabidopsis MAP65-4 plays a role in phragmoplast microtubule organization and marks the cortical cell division site.

The evolutionarily conserved MAP65 family proteins bundle anti-parallel microtubules (MTs). In Arabidopsis thaliana, mutations in the MAP65-3 gene lead to serious defects in MT organization in the phragmoplast and cause failures in cytokinesis. However, the functions of other ArabidopsisMAP65 isoforms are largely unknown. MAP65 functions were analyzed based on genetic interactions among different map65 mutations. Live-cell imaging and immunolocalization experiments revealed dynamic activities of two closely related MAP65 proteins in dividing cells. The map65-4 mutation caused synthetic lethality with map65-3 although map65-4 alone did not cause a noticeable phenotype. Furthermore, the introduction of an extra copy of the MAP65-4 gene significantly suppressed defects in cytokinesis and seedling growth caused by map65-3 because of restoring MT engagement in the spindle midzone. During mitosis, MAP65-4 first appeared at the preprophase band and persisted at the cortical division site afterwards. It was also concentrated on MTs in the spindle midzone and the phragmoplast. In the absence of MAP65-3, MAP65-4 exhibited greatly enhanced localization in the midzone of developing phragmoplast. Therefore, we have uncovered redundant but differential contributions of MAP65-3 and MAP65-4 to engaging and bundling anti-parallel MTs in the phragmoplast and disclosed a novel action of MAP65-4 at the cortical cell division site