Dieter Becker

Different roles of Enhanced Disease Susceptibility1 (EDS1) bound to and dissociated from Phytoalexin Deficient4 (PAD4) in Arabidopsis immunity

• Enhanced Disease Susceptibility1 (EDS1) is an important regulator of plant basal and receptor-triggered immunity. Arabidopsis EDS1 interacts with two related proteins, Phytoalexin Deficient4 (PAD4) and Senescence Associated Gene101 (SAG101), whose combined activities are essential for defense signaling. The different sizes and intracellular distributions of EDS1-PAD4 and EDS1-SAG101 complexes in Arabidopsis leaf tissues suggest that they perform nonredundant functions. • The nature and biological relevance of EDS1 interactions with PAD4 and SAG101 were explored using yeast three-hybrid assays, in vitro analysis of recombinant proteins purified from Escherichia coli, and characterization of Arabidopsis transgenic plants expressing an eds1 mutant (eds1(L262P) ) protein which no longer binds PAD4 but retains interaction with SAG101. • EDS1 forms molecularly distinct complexes with PAD4 or SAG101 without additional plant factors. Loss of interaction with EDS1 reduces PAD4 post-transcriptional accumulation, consistent with the EDS1 physical association stabilizing PAD4. The dissociated forms of EDS1 and PAD4 are fully competent in signaling receptor-triggered localized cell death at infection foci. By contrast, an EDS1-PAD4 complex is necessary for basal resistance involving transcriptional up-regulation of PAD4 itself and mobilization of salicylic acid defenses. • Different EDS1 and PAD4 molecular configurations have distinct and separable functions in the plant innate immune response.

Multiple genes are transcribed in Hordeum vulgare and Zea mays that carry the DNA binding domain of the myb oncoproteins

SummarycDNA clones were isolated from tissue specific cDNA libraries of barley and maize using as a probe the cDNA of the maize gene C1, a regulator of anthocyanin gene expression. C1-related homology for all of the four cDNAs characterized by sequence analysis is restricted to the N-terminal 120 amino acids of the putative proteins. This region shows striking homology to the N-proximal domain of the myb oncoproteins from vertebrates and invertebrates. Within the myb proto-oncogene family this part of the respective gene products functions as a DNA binding domain. Acidic domains are present in the C-proximal protein segments. Conservation of these sequences, together with the genetically defined regulator function of the C1 gene product, suggest that myb-related plant genes code for trans-acting factors which regulate gene expression in a given biosynthetic pathway.

Structure of a chalcone synthase gene from Hordeum vulgare

Chalcone synthase (CHS; EC 2.3.1.74) catalyses the condensation of malonyl and p-coumaroyl CoA esters to form naringenin chalcone, an aromatic C15 compound which is the first intermediate in flavonoid biosynthesis [6]. Consequently, CHS plays a central role in this branched secondary metabolite pathway leading, for example, to the formation of the anthocyanin flower pigments. The expression of the Chs gene(s) is influenced by external factors like light and regulated in a temporal and tissue-specific manner. Such regulation may occur by the recognition of cis-acting elements: for Petunia hybrida a region of the Chs gene promoter that directs flower-specific expression was recently identified [ 13]. In maize the C2 locus which encodes CHS is controlled by a variety of regulatory loci [ 3 ], four of which (B, C1, PI, R) have been shown to encode proteins with characteristics of transcriptional activators [2, 14]. These regulatory proteins are related by amino acid sequence homology to the DNAbinding domains of the MYB (C1, PI) and MYC (B, R) oncoproteins. In addition to C1 at least two other genes of yet unknown function are expressed in maize that are related to the MYB oncoprotein by the presence of the MYB DNAbinding domain [9]. In barley we have identified multiple genes with homology to Myb [9] and isolated a total of six different Myb-related cDNAs [ 11, unpublished data]. In an attempt to study the possible interaction in vivo and in vitro of any of these Myb gene products with structural genes for anthocyanin biosynthesis in barley, the genes for chalcone synthase (CHS), dihydroflavonol reductase (DFR) and UDPglucose : flavonol 3-O-glucosyltransferase (UFGT; EC 2.4.1.91) were cloned [15, unpublished data]. For one of the gene products (MYBHvl [9]) it was shown that the protein binds in vitro to a barley Chs gene and that this binding capacity is a property of the N-terminal 120 amino acids homologous to the DNA-binding domain of the animal MYB oncoproteins [8, 16]. Here we report on the nucleotide sequence of this Chs gene (Fig. 1). It contains a single intron of 1289 nucleotides which interrupts the coding sequence at the first nucleotide of a U G U codon for cysteine. An intron at this position is also present in the Chs genes from Petunia hybrida, Zea mays, Antirrhinum majus and Arabidopsis thaliana [4, 10, 12, 13]. The presumed active site cysteine Cys343 of the enzyme as postulated for chalcone synthases and resveratrol synthase [7] is located in the sequence MSSACV (Fig. 1, boxed protein sequence) highly conserved among CH S proteins from various plant species [ 10]. Within the promoter region a stretch of 17 consecutive guanosine residues (coordinates 1273-1289) is followed by direct repeats of 23 (coordinates 13101332/1443-1465) and 25 nucleotides (coordinates 1349-1373/1481-1505), respectively. The adjacent sequence G T G A T T A G (coordinates 1531-1538) is highly related to the simian virus 40 core enhancer sequence G T G G T T A G which is

The promoter of coconut foliar decay-associated circular single-stranded DNA directs phloem-specific reporter gene expression in transgenic tobacco.

A full-length double-stranded DNA copy of the single-stranded circular DNA associated with coconut foliar decay virus (CFDV) was constructed. Full-length CFDV DNA and smaller fragments were transcriptionally fused to the β-glucuronidase reporter gene and examined for promoter activity in vivo. In stably transformed tobacco plants, the CFDV DNA promoter confered a tissue-specific expression pattern in that the reporter gene was specifically expressed in the phloem tissue of the vascular system in stem, leaves and flower. These results are in agreement with the previously reported association of CFDV DNA with the phloem of its coconut host plant.

Pattern of expression of meristem-specific cDNA clones of barley (Hordeum vulgare L.)

Deoxyribonucleic-acid sequences expressed at high levels in meristematic tissues of barley (Hordeum vulgare L.) have been cloned by differential hybridization. Five out of the seven cDNA clones studied showed homologies to histone genes H2a (two clones), H2b, H3 and H4. Their patterns of expression, as studied by RNA and in-situ hybridization, were typical for genes transcribed during cell division. A sixth cDNA clone, Sab2, had a 65.7% identity (on a protein basis) to L2-like ribosomal proteins of Escherichia coli and other lower prokaryotes. In a domain of 50 amino acids, the seventh clone, Sab35, showed 69.0% sequence identity to the ribosomal protein L21 of Rattus norvegicus. The Sab35 mRNA contained in its 5′-untranslated leader sequence small open reading frames, a feature pointing to a possible translational control. The Sab35 in-situ hybridization pattern was to a certain degree different from that of the histone-like clone Sab11: it detected transcripts not only in tissues that are associated with vegetative and reproductive apices but also in sub-apical regions. The visualization in situ of transcripts coded by Sab11, 35 and 44 is discussed as a possible technique for studying differential gene expression in barley meristematic tissues.

Pathogen exploitation of an abscisic acid- and jasmonate-inducible MAPK phosphatase and its interception by Arabidopsis immunity

Significance Pathogens cause disease by deploying virulence effectors that interfere with various host targets, whereas plants counteract pathogen virulence when invoking a potent immunity known as effector-triggered immunity (ETI). Little is known about the mechanism underlying this molecular battle between plant immunity and pathogen virulence. We find that the phytohormones abscisic acid and jasmonate (JA), the signaling pathways of which are often exploited by pathogens, transcriptionally activate a common family of protein phosphatases that suppress immune-associated MAP kinases. We demonstrate that a bacterial pathogen exploits the JA-mediated suppression of MAP kinases by using a JA-mimic, whereas ETI blocks JA signaling to counteract this bacterial virulence. Our results highlight suppression and protection of MAP kinases as a molecular battle between pathogens and plants. Phytopathogens promote virulence by, for example, exploiting signaling pathways mediated by phytohormones such as abscisic acid (ABA) and jasmonate (JA). Some plants can counteract pathogen virulence by invoking a potent form of immunity called effector-triggered immunity (ETI). Here, we report that ABA and JA mediate inactivation of the immune-associated MAP kinases (MAPKs), MPK3 and MPK6, in Arabidopsis thaliana. ABA induced expression of genes encoding the protein phosphatases 2C (PP2Cs), HAI1, HAI2, and HAI3 through ABF/AREB transcription factors. These three HAI PP2Cs interacted with MPK3 and MPK6 and were required for ABA-mediated MPK3/MPK6 inactivation and immune suppression. The bacterial pathogen Pseudomonas syringae pv. tomato (Pto) DC3000 activates ABA signaling and produces a JA-mimicking phytotoxin, coronatine (COR), that promotes virulence. We found that Pto DC3000 induces HAI1 through COR-mediated activation of MYC2, a master transcription factor in JA signaling. HAI1 dephosphorylated MPK3 and MPK6 in vitro and was necessary for COR-mediated suppression of MPK3/MPK6 activation and immunity. Intriguingly, upon ETI activation, A. thaliana plants overcame the HAI1-dependent virulence of COR by blocking JA signaling. Finally, we showed conservation of induction of HAI PP2Cs by ABA and JA in other Brassicaceae species. Taken together, these results suggest that ABA and JA signaling pathways, which are hijacked by the bacterial pathogen, converge on the HAI PP2Cs that suppress activation of the immune-associated MAPKs. Also, our data unveil interception of JA-signaling activation as a host counterstrategy against the bacterial suppression of MAPKs during ETI.

An incoherent feed-forward loop mediates robustness and tunability in a plant immune network

Immune signaling networks must be tunable to alleviate fitness costs associated with immunity and, at the same time, robust against pathogen interferences. How these properties mechanistically emerge in plant immune signaling networks is poorly understood. Here, we discovered a molecular mechanism by which the model plant species Arabidopsis thaliana achieves robust and tunable immunity triggered by the microbe‐associated molecular pattern, flg22. Salicylic acid (SA) is a major plant immune signal molecule. Another signal molecule jasmonate (JA) induced expression of a gene essential for SA accumulation, EDS5. Paradoxically, JA inhibited expression of PAD4, a positive regulator of EDS5 expression. This incoherent type‐4 feed‐forward loop (I4‐FFL) enabled JA to mitigate SA accumulation in the intact network but to support it under perturbation of PAD4, thereby minimizing the negative impact of SA on fitness as well as conferring robust SA‐mediated immunity. We also present evidence for evolutionary conservation of these gene regulations in the family Brassicaceae. Our results highlight an I4‐FFL that simultaneously provides the immune network with robustness and tunability in A. thaliana and possibly in its relatives.

Cuticular wax composition in Cocos nucifera L.: physicochemical analysis of wax components and mapping of their QTLs onto the coconut molecular linkage map.

Cuticular waxes were extracted from the leaves of a coconut mapping population generated by the controlled cross of an East African Tall and a Rennell Island Tall genotype for the construction of molecular linkage maps. The wax composition was analyzed by capillary gas chromatography/mass spectrometry, and for eight of the wax compounds, their absolute and relative amounts were determined. As reported previously for a different coconut ecotype (Malayan Yellow Dwarf), lupeol methyl ether, isoskimmiwallin, and skimmiwallin were identified as the major components of coconut cuticular wax. The additional compounds were characterized as 3-β-methoxy lupane (lupane methyl ether), lupeol and the acetic acid esters of lupeol, skimmiwallinol, and isoskimmiwallinol, respectively. Minor, nonidentified compounds amounted to some 5% of total wax content and included triterpenoids, sterols, primary alcohols, and fatty acids. The variation detected for parents and progeny with respect to the wax components allowed quantitative trait locus (QTL) analyses for their biosynthetic pathways. A total of 46 QTLs could be mapped onto the coconut linkage map which was extended by amplified fragment length polymorphism and single sequence repeat markers into a high density map with more than 1,000 mapped DNA markers. Several colocated QTLs for different traits were detected reflecting the observed correlations among characters.

The Defense Phytohormone Signaling Network Enables Rapid, High-Amplitude Transcriptional Reprogramming during Effector-Triggered Immunity

The phytohormone network accelerates transcriptional reprogramming during ETI, thereby enabling rapid establishment of ETI, which is vital for effective resistance against bacterial pathogens. The phytohormone network consisting of jasmonate, ethylene, PHYTOALEXIN-DEFICIENT4, and salicylic acid signaling is required for the two modes of plant immunity, pattern-triggered immunity (PTI), and effector-triggered immunity (ETI). A previous study showed that during PTI, the transcriptional responses of over 5000 genes qualitatively depend on complex interactions between the network components. However, the role of the network in transcriptional reprogramming during ETI and whether it differs between PTI and ETI remain elusive. Here, we generated time-series RNA-sequencing data of Arabidopsis thaliana wild-type and combinatorial mutant plants deficient in components of the network upon challenge with virulent or ETI-triggering avirulent strains of the foliar bacterial pathogen Pseudomonas syringae. Resistant plants such as the wild type achieved high-amplitude transcriptional reprogramming 4 h after challenge with avirulent strains and sustained this transcriptome response. Strikingly, susceptible plants including the quadruple network mutant showed almost identical transcriptome responses to resistant plants but with several hours delay. Furthermore, gene coexpression network structure was highly conserved between the wild type and quadruple mutant. Thus, in contrast to PTI, the phytohormone network is required only for achieving high-amplitude transcriptional reprogramming within the early time window of ETI against this bacterial pathogen.