Arabidopsis triphosphate tunnel metalloenzyme2 is a negative regulator of the salicylic Acid-mediated feedback amplification loop for defense responses

Ung H, Moeder W, Yoshioka K

Plant Physiol. 2014 Oct;166(2):1009-21

PMID: 25185123

Abstract

The triphosphate tunnel metalloenzyme (TTM) superfamily represents a group of enzymes that is characterized by their ability to hydrolyze a range of tripolyphosphate substrates. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes, AtTTM1, AtTTM2, and AtTTM3. Although AtTTM3 has previously been reported to have tripolyphosphatase activity, recombinantly expressed AtTTM2 unexpectedly exhibited pyrophosphatase activity. AtTTM2 knockout mutant plants exhibit an enhanced hypersensitive response, elevated pathogen resistance against both virulent and avirulent pathogens, and elevated accumulation of salicylic acid (SA) upon infection. In addition, stronger systemic acquired resistance compared with wild-type plants was observed. These enhanced defense responses are dependent on SA, PHYTOALEXIN-DEFICIENT4, and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. Despite their enhanced pathogen resistance, ttm2 plants did not display constitutively active defense responses, suggesting that AtTTM2 is not a conventional negative regulator but a negative regulator of the amplification of defense responses. The transcriptional suppression of AtTTM2 by pathogen infection or treatment with SA or the systemic acquired resistance activator benzothiadiazole further supports this notion. Such transcriptional regulation is conserved among TTM2 orthologs in the crop plants soybean (Glycine max) and canola (Brassica napus), suggesting that TTM2 is involved in immunity in a wide variety of plant species. This indicates the possible usage of TTM2 knockout mutants for agricultural applications to generate pathogen-resistant crop plants.

Expression and gene knockdown of zebrafish Ca(2+)/calmodulin-dependent protein kinase Iδ-LL

Senga Y, Yoshioka K, Kameshita I, Sueyoshi N

Arch. Biochem. Biophys. 2013 Dec;540(1-2):41-52

PMID: 24099663

Abstract

Ca(2+)/calmodulin-dependent protein kinase Iδ (CaMKIδ) is expressed ubiquitously, but little is known about its physiological functions. Recently, we cloned and characterized two splice variants of zebrafish (Danio rerio) CaMKIδ (CaMKIδ-S/L). In the present study we cloned a new CaMKIδ isoform, CaMKIδ-LL, encoded by a different gene from CaMKIδ-S/L. While the catalytic domain of CaMKIδ-LL showed 86% identity that of CaMKIδ-S/L, it had a unique C-terminal sequence. To clarify the functional role of CaMKIδ-LL, we investigated the biological significance of this new isoform during zebrafish embryogenesis. Although CaMKIδ-LL exhibited essentially the same catalytic properties and substrate specificities as the other CaMKIδ isoforms, it showed different temporal and spatial expression. During zebrafish embryogenesis, RT-PCR analysis detected CaMKIδ-LL expression after 48 h post-fertilization. Western blotting in adult zebrafish demonstrated that CaMKIδ-LL is expressed in the brain, the eye, and, abundantly, in fins. Knockdown of CaMKIδ-LL expression using morpholino-based antisense oligonucleotides resulted in an increase in abnormal embryos with small fins and underdeveloped cartilage. These phenotypes were rescued by co-injection with recombinant CaMKIδ-LL. These results clearly indicated that CaMKIδ-LL plays an important role in the generation of cartilage and fins during zebrafish embryogenesis.

A suppressor screen of the chimeric AtCNGC11/12 reveals residues important for intersubunit interactions of cyclic nucleotide-gated ion channels

Abdel-Hamid H, Chin K, Moeder W, Shahinas D, Gupta D, Yoshioka K

Plant Physiol. 2013 Jul;162(3):1681-93

PMID: 23735507

Abstract

To investigate the structure-function relationship of plant cyclic nucleotide-gated ion channels (CNGCs), we identified a total of 29 mutant alleles of the chimeric AtCNGC11/12 gene that induces multiple defense responses in the Arabidopsis (Arabidopsis thaliana) mutant, constitutive expresser of PR genes22 (cpr22). Based on computational modeling, two new alleles, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), were identified as counterparts of human CNGA3 (a human CNGC) mutants. Both mutants lost all cpr22-mediated phenotypes. Transient expression in Nicotiana benthamiana as well as functional complementation in yeast (Saccharomyces cerevisiae) showed that both AtCNGC11/12:G459R and AtCNGC11/12:R381H have alterations in their channel function. Site-directed mutagenesis coupled with fast-protein liquid chromatography using recombinantly expressed C-terminal peptides indicated that both mutations significantly influence subunit stoichiometry to form multimeric channels. This observation was confirmed by bimolecular fluorescence complementation in planta. Taken together, we have identified two residues that are likely important for subunit interaction for plant CNGCs and likely for animal CNGCs as well.