Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen- and wound-induced signaling

Abstract

Pathogen-inducible plant promoters contain multiple cis-acting elements, only some of which may contribute to pathogen inducibility. Therefore, we made defined synthetic promoters containing tetramers of only a single type of element and present evidence that a range of cis-acting elements (boxes W1, W2, GCC, JERE, S, Gst1, and D) can mediate local gene expression in planta after pathogen attack. The expression patterns of the promoters were monitored during interactions with a number of pathogens, including compatible, incompatible, and nonhost interactions. Interestingly, there were major differences in the inducibilities of the various promoters with the pathogens tested as well as differences in the speed of induction and in the basal expression levels. We also show that defense signaling is largely conserved across species boundaries at the cis-acting element level. Many of these promoters also direct local wound-induced expression, and this provides evidence for the convergence of resistance gene, nonhost, and wound responses at the level of the promoter elements. We have used these cis-acting elements to construct improved synthetic promoters and show the effects of varying the number, order, and spacing of such elements. These promoters are valuable additions to the study of signaling and transcriptional activation during plant-pathogen interactions.

Figure 1.

Elicitor-Inducible Synthetic Plant Promoters. (A) Scheme of the synthetic promoters. Elements were inserted between the SpeI and XbaI sites in pBT10 upstream of the −46 35S promoter of Cauliflower mosaic virus (CaMV 35S). pAnos, nos terminator. (B) Sequence of the elicitor-inducible elements. Core sequences are shown underlined and in boldface. (C) Elicitor inducibility of the synthetic promoters in a parsley transient expression system. Gray bars represent GUS activity 8 hr after pep25 addition. White bars show the level of GUS activity in the absence of pep25. The fold inducibility is shown, and error bars indicate ±sem. Qualitatively similar results were obtained upon normalization with a constitutively expressed Petroselinum crispum UBI4/2 ::luciferase construct (Sprenger-Haussels and Weisshaar, 2000).

Figure 2.

Elicitor-Inducible Promoters Containing GCC-like Elements. (A) Sequence of the elicitor-inducible GCC-like elements. Core sequences are shown underlined and in boldface. (B) Elicitor inducibility of synthetic promoters containing GCC-like elements in a parsley transient expression system. Gray bars represent GUS activity 8 hr after pep25 addition. White bars show the level of GUS activity in the absence of pep25. The fold inducibility is shown, and error bars indicate ±sem. Qualitatively similar results were obtained upon normalization with a constitutively expressed P. crispum UBI4/2:: luciferase construct (Sprenger-Haussels and Weisshaar, 2000).

Figure 3.

Expression Patterns of Synthetic Promoters in Planta and in Response to Wounding. (A) Expression patterns (GUS activity) in untreated excised leaves. (B) Expression patterns 1 hr after wounding by cutting. (C) Local wound-induced expression from 4 × GCC 1 hr after wounding. (D) Local wound-induced expression from 4 × S 1 hr after wounding.

Figure 4.

Local Expression in Response to P. parasitica pv Cala2. (A) Local GUS expression 2 days after P. parasitica infection in Arabidopsis plants containing the synthetic promoter 4 × W1. Blue spots represent infection sites. (B) Local expression in 4 × GCC plants. (C) Local expression in 4 × S plants. (D) Local expression in 4 × Gst1 plants.

Figure 5.

Local Expression during Nonhost and Compatible Powdery Mildew Interactions. (A) Light micrograph of one leaf infection site on a 4 × S plant viewed at two different planes of focus. The epidermal plane (right) shows the inducing penetration attempt by a germinated B. graminis spore (sp). The differentiation of an appressorial germ tube (agt) coincides with local cell wall thickening (pap). Focusing deeper into the tissue (mesophyll plane; left) reveals GUS expression in mesophyll cells just underneath the penetration attempt. The photograph was taken 2 days after inoculation with the nonhost pathogen B. graminis. Staining of the fungus was with Coomassie blue. (B) Local expression in a 2 × W2/2 × S/2 × D plant upon B. graminis challenge. The rare successful penetration event at left triggered a cell death response (cd), whereas early aborted penetration attempts correlated with papilla formation (pap). (C) Local expression 7 days after infection with the compatible powdery mildew E. cichoracearum in a 4 × S plant. Blue spots represent infection sites. (D) Closeup of the border region of an infection site from (C). Reporter gene expression coincides with superficial mycelium (sm).

Figure 6.

Expression during Interactions with P. syringae. Using a syringe, leaves were inoculated with either isolate DC3000 (compatible interaction) or isolate DC3000 carrying the avrRpm1 gene (incompatible interaction) and were harvested after 6 or 24 hr. As controls, untreated leaves and leaves 24 hr after treatment with MgCl₂ buffer were harvested and stained for GUS activity. (A) 4 × W2. (B) 4 × JERE. (C) 4 × D. (D) 4 × GCC.

Figure 7.

Effect of Element Number on Strength and Inducibility. (A) Elicitor inducibility of synthetic promoters containing increasing numbers of the elicitor-inducible cis-acting element box W2. Gray bars represent GUS activity 8 hr after pep25 addition. White bars show the level of GUS activity in the absence of pep25. The fold inducibility is shown, and error bars indicate ±sem. (B) and (C) Expression patterns with plants containing the synthetic promoters 4 × S (B) and 2 × S (C) during interactions with P. syringae. Leaves were inoculated with either isolate DC3000 (compatible interaction) or isolate DC3000 carrying the avrRpm1 gene (incompatible interaction) and were harvested after 6 or 24 hr.

Figure 8.

Spacing Effects and Promoter Strength. (A) Sequences of the different versions of box W1 and box W2. W box core sequences are indicated in boldface. The seven additional bases in box W1 new are shown in boldface and underlined. The seven unrelated bases in box W1 5 prime mut are shown in boldface italics and underlined. (B) Elicitor inducibility of the synthetic promoters shown in (A). Gray bars represent GUS activity 8 hr after pep25 addition. White bars show the level of GUS activity in the absence of pep25. The fold inducibility is shown, and error bars indicate ±sem. (C) Elicitor inducibility of synthetic promoters with elements inserted at different positions. Elements were inserted into either the SpeI site (−74) or the XbaI site (−56).

Figure 9.

Box D. (A) Sequences of the different versions of box D. The six additional bases in box D are shown in boldface and underlined. The six unrelated bases in box D mut and 4 × D 3 prime mut are shown in boldface italics and underlined. (B) Elicitor inducibility of the synthetic promoters shown in (A). Gray bars represent GUS activity 8 hr after pep25 addition. White bars show the level of GUS activity in the absence of pep25. The fold inducibility is shown, and error bars indicate ±sem.

Figure 10.

Synthetic Promoters Containing Combinations of Elements. (A) Elicitor inducibility of synthetic promoters containing combinations of box W2 and box S. Gray bars represent GUS activity 8 hr after pep25 addition. White bars show the level of GUS activity in the absence of pep25. The fold inducibility is shown, and error bars indicate ±sem. (B) GUS expression pattern of the synthetic promoter 2 × W2/2 × S/2 × D 3 days after treatment with B. graminis (nonhost interaction). (C) GUS expression pattern of the synthetic promoter 4 × W2/4 × S 6 and 12 hr after treatment with P. syringae carrying the avrRpm1 gene (incompatible interaction).