The fas operon of Rhodococcus fascians encodes new genes required for efficient fasciation of host plants

Abstract

Three virulence loci (fas, ftt, and hyp) of Rhodococcus fascians D188 have been identified on a 200-kb conjugative linear plasmid (pFiD188). The fas locus was delimited to a 6.5-kb DNA fragment by insertion mutagenesis, single homologous disruptive recombination, and in trans complementation of different avirulent insertion mutants. The locus is arranged as a large operon containing six open reading frames whose expression is specifically induced during the interaction with host plants. One predicted protein is homologous to P-450 cytochromes from actinomycetes. The putative ferredoxin component is of a novel type containing additional domains homologous to transketolases from chemoautotrophic, photosynthetic, and methylotrophic microorganisms. Genetic analysis revealed thatfas encodes, in addition to the previously identified ipt, at least two new genes that are involved in fasciation development, one of which is only required on older tobacco plants.The interaction between Rhodococcus fascians and host plants leads to the loss of apical dominance and the development of multiple malformed shoots at the site of infection (fasciation). This proliferation is governed by multiple bacterial loci located both on a linear Fi plasmid and on the chromosome (reference 8 and unpublished results). From the three identified loci on pFiD188, only inactivation of one (fas) leads to a complete loss of phytopathogenicity. In this locus, an open reading frame (ORF) coding for an isopentenyltransferase (ipt) has been identified by sequence homology to other cytokinin biosynthesis genes and by biochemical approaches (8). Expression of ipt in R. fascians is regulated by an inducing factor which can only be detected in significant amounts in extracts from tumor tissues (induced by R. fascians) (8).In this paper, we present genetic data that allowed the definition of the fas locus on a 6.5-kb DNA fragment whose complete nucleotide sequence was determined. Six ORFs directed in the same orientation as the previously defined ipt were identified. ORFi and the amino-terminal part of ORF2 have extensive homologies, to P-450 cytochromes and ferredoxins from actinomycetes, whereas the carboxy-terminal portion of ORF2 has conserved regions homologous to the aminoterminal part of transketolases from chemoautotrophic, photosynthetic, or methylotrophic microorganisms.The isolation of new fas mutants and complementation analysis revealed that fas encodes new genes involved in fasciation of the host plant.MATERIALS AND METHODS Bacterial strains and plasmids. Growth conditions and antibiotic concentrations for R. fascians strains have been described previously (11). The Escherichia coli strains used for

Degradation of the thiocarbamate herbicide EPTC (S-ethyl dipropylcarbamothioate) and biosafening by Rhodococcus sp. strain NI86/21 involve an inducible cytochrome P-450 system and aldehyde dehydrogenase

“…In S. griseolus, the structural genes for the ferredoxins of the [3Fe-4S] type are located downstream of their corresponding cytochrome P-450 genes, but there is no evidence for linkage to a ferredoxin reductase gene (55,59). A similar gene organization has been described for the cytochrome P-450 induced in Streptomyces griseus growing in medium with soybean flour (78) and for the fasciation-related cytochrome P-450 with unknown function located on the pFiD188 plasmid of R. fascians (9). The cytochrome P-450 systems induced in R. rhodochrous strains by n-octane (70) or alkoxyphenols and alkoxybenzoates (18,37) and in Rhodococcus chlorophenolicus by pentachlorophenol (80) have not yet been genetically characterized.…”

“…A multiple alignment of available sequences for bacterial cytochrome P450s revealed that the Rhodococcus gene represents the first member of a new family, CYP116 (54). The cytochrome P-450 from the fas operon of Rhodococcus fascians, which is required for efficient fasciation of host plants (9), bears little homology with the EPTC-induced cytochrome P-450 from Rhodococcus sp. strain NI86/21.…”

Cytokinins Mediate Resistance againstPseudomonas syringaein Tobacco through Increased Antimicrobial Phytoalexin Synthesis Independent of Salicylic Acid Signaling

“…CKs are also produced by a range of various microbial pathogens, including Pseudomonas syringae (Akiyoshi et al, 1987;Morris et al, 1991) and leaf-mining insects (Engelbrecht, 1968), causing the formation of green islands, galls, growth abnormalities, and manipulation of primary carbon metabolism (Balibrea Lara et al, 2004). For several pathogens, it was shown that this CK production is essential for infection (Crespi et al, 1994;Hwang et al, 2010). Based on the induction of sink metabolism by CKs Walters et al, 2008), it has been suggested that the host physiology is altered in response to CKs to allow the pathogen maximum access to nutrients early in (hemi)biotrophic interactions (Walters et al, 2008).…”

Chromosomal Locus That Affects Pathogenicity ofRhodococcus fascians

“…The fas locus contains six genes that encode an isopentenyltransferase (IPT) and enzymes involved in the modification of the IPT product. Together they form the machinery for the synthesis of a signal molecule that is essential for symptom development (7,8,48,49). The expression of the fas genes is controlled through a complex network encompassing both transcriptional and translational regulation (50).…”