The Elicitor-Inducible Alfalfa Isoflavone Reductase Promoter Confers Different Patterns of Developmental Expression in Homologous and Heterologous Transgenic Plants

Abstract

In legumes, the synthesis of infection- and elicitor-inducible antimicrobial phytoalexins occurs via the isoflavonoid branch of the phenylpropanoid pathway. To study transcriptional regulation of isoflavonoid pathway-specific genes, we have isolated the gene encoding isoflavone reductase (IFR), which is the enzyme that catalyzes the penultimate step in the synthesis of the phytoalexin medicarpin in alfalfa. Chimeric gene fusions were constructed between 765- and 436-bp promoter fragments of the IFR gene and the beta-glucuronidase reporter gene and transferred to alfalfa and tobacco by Agrobacterium-mediated transformation. Both promoter fragments conferred elicitor-mediated expression in cell suspension cultures derived from transgenic plants of both species and fungal infection-mediated expression in leaves of transgenic alfalfa. Developmental expression directed by both promoter fragments in transgenic alfalfa was observed only in the root meristem, cortex, and nodules, which is consistent with the accumulation of endogenous IFR transcripts. However, in transgenic tobacco, expression from the 765-bp promoter was observed in vegetative tissues (root meristem and cortex, inner vascular tissue of stems and petioles, leaf tips, and stem peripheries adjacent to petioles) and in reproductive tissues (stigma, placenta, base of the ovary, receptacle, seed, tapetal layer, and pollen grains), whereas the 436-bp promoter was expressed only in fruits, seed, and pollen. These data indicate that infection/elicitor inducibility of the IFR promoter in both species and developmental expression in alfalfa are determined by sequences downstream of position -436, whereas sequences between -436 and -765 confer a complex pattern of strong ectopic developmental expression in the heterologous species that lacks the isoflavonoid pathway.

Regulation of Sesquiterpene Cyclase Gene Expression (Characterization of an Elicitor- and Pathogen-Inducible Promoter)

“…A P-box element initially identified by in vivo footprinting (Lois et al, 1989) and subsequent isolation of BPF-1, a P-box-binding protein (da Costa e Silva et al, 1993), is conserved in sequence and position within several PAL genes in parsley (Hahlbrock et al, 1995). Although no sequence-specific motifs have yet been specified within the alfalfa IFR promoter, 400 bp of the TATA-proximal portion of this promoter was sufficient to direct proper developmental and elicitor-inducible expression of the GUS gene in transgenic alfalfa (Oommen et al, 1994).…”

“…Among these, promoters for the CHS15 gene from bean (Dron, 1988;Harrison, 1991), the PAL-l (Lois et al, 1989) and PR2 (van de Locht et al, 1990) genes from parsley, a PRlOa gene from potato (Despres et al, 1995), an IFR gene from alfalfa (Oommen et al, 1994), and the str246c (Gough et al, 1995) and several PR Eyal et a\., 1993;Shinshi et al, 1995) genes from tobacco have been examined in detail for cis-sequences controlling gene expression patterns. The CHS15 promoter, for example, contains G-and H-box elements that have been correlated with defense gene expression as well as proper developmental expression of the CHS gene in flowers and root tissues.…”

“…GST is induced by H,O, (Levine et al, 1994), PAL and other phenylpropanoid biosynthetic genes are activated by MJ (Ellard-Ivey and Douglas, 1996), and many of the PR genes are defined by their SA inducibility (Ward et al, 1991;Ryals et al, 1994 some tissues o r cell types. The IFR promoter directs strong expression i n root meristem, cortex, a n d nodules of transgenic alfalfa plants (Oommen et al, 1994), whereas the str246c promoter directs very high levels of root a n d sepal expression in transgenic plants (Gough et al, 1995). Except for a low leve1 of wound-inducible expression, we have not detected any EAS4 promoter-directed GUS expression in any plant tissues, including flowers and a11 reproductive structures (S. Yin and J. Chappell, unpublished data), throughout plant development other than in those challenged wíth elicitors or pathogens.…”

“…6) was compared with several other defense gene promoters, such as those for PAL (Lois et al, 1989), CHS (Harrison et al, 1991), IFR (Oommen et al, 1994), PR1 , and PR2 (van de Locht et al, 1990), for putative regulatory motifs, and other sequences. The EAS4 promoter does not contain any sequences similar to the P-box (CCANCNA-ACNCC) or H-box (CCTACCN,CT), cis-elements that have been found in several PAL and CHS promoters and implicated in elicitor inducibility (Lois et al, 1989;Yu et al, 1993;Dixon and Paiva, 1995;Hahlbrock et al, 1995), but does contain sequences that are similar to the G-box (-366) (Weisshaar et al, 1991;Schindler et al, 1992;Williams et al, 1992), the as-1 (-195) (Katagiri et al, 1989;Lam et al, 1989), and the myb-box (-94) (Biedenkapp et al, 1988;Baranowskij et al, 1994).…”

Induction of Microbial Genes for Pathogenesis and Symbiosis by Chemicals from Root Border Cells

“…It has been known for nearly a century that root exudates-the chemicals released from root systems into the soil-have profound effects on microorganisms in the rhizosphere (for review, see Curl and Truelove, 1986;Rovira, 1991). In recent years, specific sugars and phenolic compounds from plant exudates have been shown to induce microbial genes required for pathogenesis and symbiosis (Rossen et al, 1985;Stachel et al, 1985;Shearman et al, 1986;Peters and Long, 1988), and the cellular sources of such chemicals are now being examined (Maxwell and Phillips, 1990;Graham, 1991;Oommen et al, 1994;McKhann et al, 1997).…”