Purification and biochemical characterization of proteins which bind to the H-box cis-element implicated in transcriptional activation of plant defense genes

Abstract

The H-box (CCTACC(N)7CT(N)4A), which occurs three times within the -154 to -42 region of the bean chalcone synthase chs15 promoter, is important for developmental regulation of chs15, and induction of chs15 and coordinately regulated defense genes by elicitors and other stress stimuli. Two protein factors, KAP-1 and KAP-2, which recognize conserved features in the H-box motif, were purified from bean cell suspension cultures by a combination of ion exchange chromatography and DNA affinity chromatography. KAP-1 is a 97 kDa polypeptide, whereas KAP-2 comprises two polypeptides of 76 and 56 kDa. KAP-1 and KAP-2 also differ in the sensitivity of their DNA-bound forms to trypsin. Dephosphorylation of KAP-1 or KAP-2 affects the mobility of the protein/H-box binding complex in gel shift assays but does not inhibit DNA binding. Elicitation of bean cell suspensions with glutathione does not affect the total cellular activities of KAP-1 or KAP-2, but causes a rapid increase in the specific activities of both factors in the nuclear fraction, consistent with a role for these factors in the signal pathway for elicitor induction of chs15 and related defense genes.

Two cold-inducible genes encoding lipid transfer protein LTP4 from barley show differential responses to bacterial pathogens

“…Under the same conditions, no significant activity was driven by the endospermspecific promoter from barley Itrl gene, which was used as a negative control. Activities of the two Ltp gene constructions found in different tissues, using the Ubl-LUC chimeric fusión as internal control, were similar to each other and essentially reproduced the expression pattern obtained by Northern blot analysis, except that Guiltinan et al 1990;Schindler et al 1992 Lois et al 1989;Cramer et al 1989;Ohl et al 1990;Loake et al 1992;Yu et al 1993); and AP-1, consensus TGACACA (API; Després et al 1995). The alignment was constructed out using the CLUSTAL W programme (Thompson et al 1994), and the SIGNAL SCAN 4.0 programme (Prestridge 1991) was used to search for sequence motifs Líp4.2 was more active than Ltp4.3 in endosperm, and Ltp4.3 was active in roots, while Ltp4.2 was not (Fig.…”

“…IB). The first motif is present in promoters of phenylpropanoid biosynthetic genes (Cramer et al 1989;Lois et al 1989;Ohl et al 1990;Loake et al 1992;Yu et al 1993), which have been shown to display elicitor-inducible and lightinducible footprints in vivo (Lois et al 1989). The presence of H-box and G-box motifs in the promoter of these genes has been found to be required for elicitor induction (Loake et al 1992).…”

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

“…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). The EAS4 promoter does not contain an ethylene-responsive GCC-box, which is conserved in a number of tobacco pathogenesis-related protein genes (Ohme and Shinshi, 1990;Eyal et al, 1993;Hart et al, 1993;Shinshi et al, 1995).…”

Phosphorylation of Opaque2 changes diurnally and impacts its DNA binding activity.

“…Other studies have also reported in vivo phosphorylation of such factors in plants, but the protein isoforms have never been characterized (Datta and Cashmore, 1989;Harrison et al, 1991;Klimczak et al, 1992;Sarokin and Chua, 1992;Sun et al, 1993;Yu et al, 1993;Harter et al, 1994;Tjaden and Coruzzi, 1994;Després et al, 1995;Klimczak et al, 1995).…”