Cloning of the gene for indoleacetic acid-lysine synthetase from Pseudomonas syringae subsp. savastanoi

Abstract

The phytopathogen Pseudomonas syringae subsp. savastanoi incites the production of galls on olive and oleander plants. Gall formation is dependent on bacterial production of the phytohormone indoleacetic acid (IAA). The genetic determinants for IAA synthesis are located on a plasmid (pIAA) and are organized in an operon in oleander strains of the bacterium. P. syringae subsp. savastanoi further converts IAA to an amino acid conjugate, 3-inidole-acetyl-r-L-lysine (IAA-lysine). The gene for IAA-lysine synthetase (iaaL) was found on the IAA plasmid by screening pIAA deletion mutants for the ability to convert IAA to IAA-lysine. The iaaL locus was then cloned in the vector pUC8 from a bank of P. syringae subsp. savastanoi EW2009 plasmid DNA to construct recombinant plasmid pLG87. The specific activity of IAA-lysine synthetase in Escherichia coli transformed with pLG87 was 47 times higher than that of the enzyme extract from P. syringae subsp. savastanoi. The direction of transcription of the iaaL gene was determined to be opposite to that of the IAA operon. The location of the iaaL gene on pIAAl was mapped by TnS insertion mutagenesis to a 2.5-kilobasepair fragment 2 kilobase pairs from the IAA operon.Pseudomonas syringae subsp. savastanoi (Smith) Stevens incites a disease of oleander (Nerium oleander L.) and olive (Olea europa L.) that is characterized by tumorous outgrowths on the stems and leaves of infected plants (21,26,27). This development of galls is dependent on bacterial production of the phytohormone indoleacetic acid (IAA) (6, 23). The two enzymes involved in the conversion of tryptophan to IAA are tryptophan monooxygenase, which converts tryptophan to indoleacetamide, and indoleacetamide hydrolase, which catalyzes the conversion of indoleacetamide to IAA (16,17). IAA can be further metabolized to an amino acid conjugate, 3-indole-acetyl-e-L-lysine (IAA-lysine) (12, 13).The genes for tryptophan monooxygenase, iaaM, and indoleacetamide hydrolase, iaaH, are organized in an operon in P. syringae subsp. savastanoi (7,8,28); the iaaM locus is the first gene transcribed. In strains isolated from oleander galls, the IAA operon is located on a plasmid (pIAA) (6, 9). Oleander gall isolates selected for resistance to the tryptophan analog a-methyl tryptophan are often incapable of synthesizing IAA and cannot incite the production of galls when inoculated onto oleander plants (23). These avirulent IAA-mutants fall into two classes when surveyed for their plasmid composition: those that lack the IAA operon, either by the loss of the IAA plasmid or by deletion of the IAA genes, and those that contain insertions in the iaaM gene (6,8,9).Certain isolates of P. syringae subsp. savastanoi convert IAA to 13

Indole-3-acetic acid in plant–pathogen interactions: a key molecule for in planta bacterial virulence and fitness

“…tomato DC3000 [30,64,65]. However, opposite results for P. savastanoi could likely be due to downstream transcriptional consequences derived from inactivation of the iaaL gene by transposon mutagenesis [30]. Concerning P. syringae pv.…”

“…The best characterized Trp-dependent pathway in bacteria is the two-step process denoted as the indole-3-acetamide (IAM) pathway, where the enzymes tryptophan-2-monooxygenase (IaaM) and IAM hydrolase (IaaH) are encoded by the iaaM and iaaH genes, respectively, and sequentially convert Trp to IAM and then to IAA. This is the most common pathway in phytopathogenic bacteria, including P. savastanoi, in which the presence of the iaaL gene encoding the enzyme that conjugates IAA to the amino acid lysine to give IAAeLys was first demonstrated [30]. This gene is widely distributed and conserved among P. syringae sensu lato species and pathovars, and no significant homology with any plant IAA-conjugating enzyme has been found [31].…”

Auxin Biosynthesis

“…Auxin concentrations in cells or tissues can now be increased or decreased by regulating auxin biosynthesis in plants. Auxin conjugation and degradation can also be manipulated to alter auxin concentrations in plants (Glass and Kosuge, 1986;Peer et al, 2013;Pencik et al, 2013;Zhao et al, 2013). In this chapter, I focus on discussing two general strategies to control auxin biosynthesis in plants: 1) use auxin biosynthesis inhibition by chemicals; 2) genetic activation or deactivation of auxin biosynthetic genes.…”

The MossPhyscomitrella patensReproductive Organ Development Is Highly Organized, Affected by the TwoSHI/STYGenes and by the Level of Active Auxin in theSHI/STYExpression Domain

“…To test if the arrest in Ppshi archegonia development could be a result of reduced auxin levels in PpSHI expression domains, we produced moss lines expressing the Pseudomonas syringae subspecies savastanoi indole-3-acetic acid-lysine synthetase (iaaL) gene (Glass and Kosuge, 1986;Romano et al, 1991), encoding an auxin-conjugating enzyme converting free auxin to inactive Lys conjugates, from the PpSHI2 promoter. Two out of five verified transgenic lines showed phenotypic deviations from the wild type, and the severity of the deviations correlated with the expression level of PpSHI2 pro :IAAL, analyzed by real-time quantitative PCR ( Fig.…”