Ole Mattsson

Arabidopsis MAP Kinase 4 Negatively Regulates Systemic Acquired Resistance

Transposon inactivation of Arabidopsis MAP kinase 4 produced the mpk4 mutant exhibiting constitutive systemic acquired resistance (SAR) including elevated salicylic acid (SA) levels, increased resistance to virulent pathogens, and constitutive pathogenesis-related gene expression shown by Northern and microarray hybridizations. MPK4 kinase activity is required to repress SAR, as an inactive MPK4 form failed to complement mpk4. Analysis of mpk4 expressing the SA hydroxylase NahG and of mpk4/npr1 double mutants indicated that SAR expression in mpk4 is dependent upon elevated SA levels but is independent of NPR1. PDF1.2 and THI2.1 gene induction by jasmonate was blocked in mpk4 expressing NahG, suggesting that MPK4 is required for jasmonic acid-responsive gene expression.

The MAP kinase substrate MKS1 is a regulator of plant defense responses

Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)‐dependent resistance and for activation of jasmonate (JA)‐dependent defense gene expression. To understand MPK4 signaling mechanisms, we used yeast two‐hybrid screening to identify the MPK4 substrate MKS1. Analyses of transgenic plants and genome‐wide transcript profiling indicated that MKS1 is required for full SA‐dependent resistance in mpk4 mutants, and that overexpression of MKS1 in wild‐type plants is sufficient to activate SA‐dependent resistance, but does not interfere with induction of a defense gene by JA. Further yeast two‐hybrid screening revealed that MKS1 interacts with the WRKY transcription factors WRKY25 and WRKY33. WRKY25 and WRKY33 were shown to be in vitro substrates of MPK4, and a wrky33 knockout mutant was found to exhibit increased expression of the SA‐related defense gene PR1. MKS1 may therefore contribute to MPK4‐regulated defense activation by coupling the kinase to specific WRKY transcription factors.

Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus

Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4–wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

Autophagic Components Contribute to Hypersensitive Cell Death in Arabidopsis

Autophagy has been implicated as a prosurvival mechanism to restrict programmed cell death (PCD) associated with the pathogen-triggered hypersensitive response (HR) during plant innate immunity. This model is based on the observation that HR lesions spread in plants with reduced autophagy gene expression. Here, we examined receptor-mediated HR PCD responses in autophagy-deficient Arabidopsis knockout mutants (atg), and show that infection-induced lesions are contained in atg mutants. We also provide evidence that HR cell death initiated via Toll/Interleukin-1 (TIR)-type immune receptors through the defense regulator EDS1 is suppressed in atg mutants. Furthermore, we demonstrate that PCD triggered by coiled-coil (CC)-type immune receptors via NDR1 is either autophagy-independent or engages autophagic components with cathepsins and other unidentified cell death mediators. Thus, autophagic cell death contributes to HR PCD and can function in parallel with other prodeath pathways.

The Arabidopsis lue1 mutant defines a katanin p60 ortholog involved in hormonal control of microtubule orientation during cell growth

The lue1 mutant was previously isolated in a bio-imaging screen for Arabidopsis mutants exhibiting inappropriate regulation of an AtGA20ox1 promoter-luciferase reporter fusion. Here we show that lue1 is allelic to fra2, bot1 and erh3, and encodes a truncated katanin-like microtubule-severing protein (AtKSS). Complementation of lue1 with the wild-type AtKSS gene restored both wild-type stature and luciferase reporter levels. Hormonal responses of lue1 to ethylene and gibberellins revealed inappropriate cortical microtubule reorientation during cell growth. Moreover, a fusion between the AtKSS protein and GFP decorated cortical microtubules. A yeast two-hybrid screen with AtKSS as the bait identified proteins related to those involved in microtubule processing, including a katanin p80 subunit and a kinesin ortholog. These results indicate that AtKSS is involved in microtubule dynamics in response to plant hormones.

An Arabidopsis callose synthase

Abstractβ-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic β-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant β-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce β-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast β-1,3-glucan synthase whose expression partially complements a yeast β-1,3-glucan synthase mutant. AtGsl5 is developmentally expressed at highest levels in flowers, consistent with flowers having high β-1,3-glucan synthase activities for deposition of callose in pollen. A role for AtGsl5 in callose synthesis is also indicated by AtGsl5expression in the Arabidopsismpk4 mutant which exhibits systemic acquired resistance (SAR), elevated β-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5mRNA accumulation is induced by SA in wild-type plants, while expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant.

Detection, expression and specific elimination of endogenous β-glucuronidase activity in transgenic and non-transgenic plants

Abstract With the aim of making a system using the GUS gene as a selection gene, the effects of two toxin glucoronides were tested on transgenic tobacco cells ( Nicotiana tabacum L.) containing a β-glucuronidase (GUS) gene from Escherichia coli and on non-transgenic cells. No significant difference in toxicity was observed between transgenic and non-transgenic cells. We found that this most probably was due to the activity of an endogeneous GUS enzyme, which could be detected in all plant species tested, e.g. tobacco, sugar beet ( Beta vulgaris L.), oilseed rape ( Brassica napus L.), pea ( Pisum sativum L.), wheat ( Triticum sativum L.) and rhubarb ( Rheum rhaponticum L.) This indicates that GUS may be ubiquitous to plants contrary to earlier assmptions. The endogenous enzyme is active at pH 4–5 and the activity is eliminated without reducing the introduced GUS activity when pH is elevated. In addition the endogenous GuS can be selectively inhibited at high temperatures. Modifications according to these findings can be employed in standard GUS assays to avoid misinterpretations when the expression of tissue specific promoters is tested.

Arabidopsis VARIEGATED 3 encodes a chloroplast- targeted, zinc-finger protein required for chloroplast and palisade cell development

The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3 protein containing novel repeats and zinc fingers described as protein interaction domains. VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma. Metabolic profiling demonstrates that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development.

Employment of hydrolytic enzymes in the study of the level of DNA methylation.

A new method for the determination of the level of DNA methylation was established. The method involves enzymatic hydrolysis of DNA by nuclease P1 and bacterial alkaline phosphatase, and separation of the resulting deoxyribonucleosides by HPLC. By this method, DNA was hydrolysed completely to the five deoxyribonucleosides and the complete base composition was determined. Pairing bases were shown to occur in similar amounts, and analysis could be performed on as little as 1 microgram of DNA with a high degree of reproducibility. Among other enzymes hitherto used in order to hydrolyze DNA, micrococcal nuclease, phosphodiesterase II and nuclease P1 have been shown to cause deamination of deoxyadenosine, while deoxyribonuclease I, phosphodiesterase I and bacterial alkaline phosphatase have been shown to be sensitive to contamination by RNA, and to release 5-methyldeoxycytidine at a slower rate than the other four deoxyribonucleosides. Neither of these effects was seen with the new method.

Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture

The predominant peroxidase (pl 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely sequenced. The cDNA clone comprised 1194 bp and encodes a 30 residue signal peptide and a 305 residue mature protein (M r 31 966). The sequence of the mature protein is 95% identical to the well‐characterized horseradish peroxidase HRP A2 and has therefore been designated ATP A2. Three introns at positions identical to those found in Arabidopsis and horseradish genes encoding cationic peroxidases were identified. RT‐PCR analysis revealed root‐specific expression.