Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem

Differential expression of two calmodulin genes in response to physical and chemical stimuli

Two different calmodulin (CaM) cDNAs (MBCaM-1 and MBCaM-2) were isolated from a vigna radiata λgt 11 library by screening with a heterologous Arabidopsis cDNA probe (TCH-1). Both cDNAs are 85% homologous inside the coding region but are highly divergent outside this region. The polypeptides encoded by MBCaM-1 and MBCaM-2 are identical except for two conservative substitutions at positions 7 and 10. Southern analysis revealed that both cDNAs are encoded by different genes. Expression studies revealed different patterns of expression of both genes. MBCaM-1 mRNA exhibited a dramatic transient increase in response to touch, while MBCaM-2 expression showed a steady but small increase as compared to MBCaM-1. When plants were grown in complete darkness MBCaM-1 was undetectable and MBCaM-2 exhibited very low levels of expression. One hour after exposure of etiolated seedlings to light MBCaM-1 showed no change, while MBCaM-2 expression was increased. After a 6 h exposure to light there was an induction of both MBCaM-1 and MBCaM-2; however, the magnitude of this increase was much greater for MBCaM-2. When plants were grown under a 16 h light/8 h dark cycle the mRNA levels for MBCaM-1 were lower during the light period and increased during the beginning of the night cycle, while MBCaM-2 showed no change. Plants treated with indole-3-acetic acid had a peak in MBCaM-1 expression 6 h after treatment initiation with a slight decline 3 h after the peak, while MBCaM-2 showed a steady but small increase over time as compared to MBCaM-1. When plants were subjected to salt stress they showed an increase in MBCaM-1 expression 2 h after treatment initiation reaching a maximum after 4 h with no further increase after 6 h, while MBCaM-2 remained unchanged over the time course

Calcium-dependent protein kinase gene expression in response to physical and chemical stimuli in mungbean (Vigna radiata)

Protein kinases are important in eukaryotic signal transduction pathways. In this study we designed degenerate oligonucleotides corresponding to two conserved regions of protein kinases and using the polymerase chain reaction (PCR) have amplified a 141 bp fragment of DNA from mungbeans (Vigna radiata Rwilcz cv. Berken). Sequence analysis of the PCR products indicates that they encode several putative protein kinases with respect to their identity with other known plant protein kinases. Using one of the six fragments (CPK3-8), we isolated a 2022 bp cDNA (VrCDPK-1) from a Vigna radiata lambda gt11 library. Vr CDPK-1 has a 96 bp 5'-untranslated region and a 465 bp 3'-untranslated region and an open reading frame of 1461 bp. VrCDPK-1 contains all of the conserved regions commonly found in calcium dependent protein kinases (CDPK). VrCDPK-1 shares 24 to 89% sequence identity with previously reported sequences for plant CDPKs at the protein level. Southern analysis revealed the presence of several copies of the CDPK gene. VrCDPK-1 expression was stimulated when mungbean cuttings were treated with CaCl2, while treatment with MgCl2 had no effect. We are reporting for the first time a CDPK gene in mungbean which is inducible by mechanical strain. Cuttings treated with indole-3-acetic acid (IAA) or subjected to salt stress showed an increase in VrCDPK-1 expression. There was a dramatic stimulation in VrCDPK-1 expression 6 h after cuttings were treated with cycloheximide

Identification of two new members of the l-aminocyclopropane-l-carboxylate synthase-encoding multigene family in mung bean

The key enzyme regulating ethylene biosynthesis in higher plants is l-aminocyclopropane-l-carboxylate (ACC) synthase. In mung bean (MB), the existence of three genes encoding this enzyme has previously been reported [Botella et al., Plant Mol. Biol. 18 (1992) 793-797], one of which corresponds to a full-length indole-3-acetic acid-inducible cDNA [Botella et al., Plant Mol. Biol. (1992) 425-436], In this paper we report the cloning of two new genomic sequences coding for ACC synthase in MB (MAC-4 and MAC-5). MAC-4 is 1340 bp long and encodes 388 amino acids (aa) while MAC-5 is 1393 bp long and encodes for 391 aa. Genomic Southern analysis suggests the existence of only one copy of each gene in the genome

Identification and characterization of a full-length cDNA encoding for an auxin-induced 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments and expression of its mRNA in response to indole-3-acetic acid

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) is the key regulatory enzyme in the ethylene biosynthetic pathway. The identification and characterization of a full-length cDNA (pAIM-1) 1941 bp in length for indole-3-acetic acid (IAA)-induced ACC synthase is described in this paper. The pAIM-1 clone has an 87 bp leader and a 402 bp trailing sequence. The open reading frame is 1452 bp long encoding for a 54.6 kDa polypeptide (484 amino acids) which has a calculated isoelectric point of 6.0. In vitro transcription and translation experiments support the calculated molecular weight and show that the enzyme does not undergo processing. Eleven of the twelve amino acid residues which are conserved in aminotransferases are found in pAIM-1. The sequence for pMAC-1 which is one of the 5 genes we have identified in mung bean is contained in pAIM-1. pAIM-1 shares between 52 to 65% homology with previously reported sequences for ACC synthase at the protein level. There is little detectable pAIM-1 message found in untreated mung bean tissues; however, expression is apparent within 30 min following the addition of 10 μM IAA reaching a peak after approximately 5 h with a slight decrease in message after 12 h. These changes in message correlate with changes in ACC levels found in these tissues following treatment with 10 μM IAA