Arabidopsis RelA/SpoT homologs implicate (p)ppGpp in plant signaling

Abstract

Arabidopsis RPP5 is a member of a large class of pathogen resistance genes encoding nucleotide-binding sites and leucine-rich repeat domains. Yeast two-hybrid analysis showed that RPP5 specifically interacts with At-RSH1, an Arabidopsis RelA/SpoT homolog. In Escherichia coli, RelA and SpoT determine the level of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effector nucleotides of the bacterial stringent response. Functional analysis in E. coli and in Streptomyces coelicolor A3 (2) showed that At-RSH1 confers phenotypes associated with (p)ppGpp synthesis. We characterized two additional Arabidopsis RelA/SpoT homologs, At-RSH2 and At-RSH3. At-RSH genes may regulate a rapid plant (p)ppGpp-mediated response to pathogens and other stresses.

Figure 1

Overview of RPP5/At-RSH1 interactions and (p)ppGpp synthetase domain-containing proteins. (A) Domain structure of RPP5 and interaction of RPP5 bait constructs with At-RSH1 (RSH1–18) in the yeast two-hybrid assay. β-Galactosidase ± SD in Miller units are averages from two replicates with three transformants. (B) Domain structure of At-RSH1, hydrophilicity analysis, and the five groups with At-RSH1 clones. R5-int, RPP5 interaction domain; TM, two transmembrane segments. (C) Domain structure of At-RSH2 and At-RSH3 with the (p)ppGpp synthetase domains aligned relative to that of At-RSH1, E. coli RelA (GenBank J04039) and SpoT (M24503), and S. coelicolor RelA (X87267).

Figure 2

At-RSH1, 2, and 3 transcript analysis and gene structures. (A) Poly(A) RNA gel blot with leaf RNA isolated from the Arabidopsis Landsberg erecta (Ler) and Columbia (Col-0) ecotypes, hybridized to radiolabeled cDNA sequences corresponding to At-RSH1 and At-RSH2; transcript sizes indicated in kilobases. (B) Intron/exon structures of At-RSH1, 2, and 3. Wide rectangles indicate transcribed regions, and solid rectangles constitute coding sequence. Narrow rectangles indicate 5′ and 3′ sequences, and intron positions are marked by arrowheads.

Figure 3

Sequence relationships between the (p)ppGpp synthetase domains of At-RSH1 and At-RSH2, and those of E. coli (Ec) RelA and SpoT and S. coelicolor (Sc) RelA. At-RSH1 and S. coelicolor RelA contain ATP/GTP-binding site motifs (P-loop; boxed) that are absent in other RelA/SpoT proteins.

Figure 4

Phylogenetic analysis of the (p)ppGpp synthetase domains of members of the RelA/SpoT family. The At-RSH1, 2, and 3 homologs and the E. coli and S. coelicolor RelA/SpoT proteins are shown in boldface type. At-RSH1, 2, and 3 cluster with RelA/SpoT proteins from intracellular pathogens (underlined).

Figure 5

Functional analysis of At-RSH1 in bacteria. (A) At-RSH1 complements an E. coli relA⁻ mutant. The 1.6-kb 5′ region of At-RSH1 restores growth of the E. coli relA mutant (CF1652) on SMG medium at 30°C. (B) At-RSH1 expression is lethal in an E. coli relA⁻, spoT⁻ double mutant. The 1.6-kb 5′ region of At-RSH1 was cloned in the temperature-inducible pT7–7 vector (26). Expression was induced by rapidly transferring rich L-broth cultures from 30°C to 42°C (denoted by arrows). Induced expression of At-RSH1 (1.6 kb) in the E. coli relA mutant (CF1652) slightly reduces growth rate (Upper), whereas in the E. coli relA, spoT double mutant (CF1693) growth is abolished (Lower). (C) At-RSH1 expression in S. coelicolor M600. At-RSH1 was expressed from the thiostrepton-inducible tipA promoter of pIJ8600 (28). Spores of each strain were dropped on SMMS medium (28) and allowed to dry. Twelve microliters of 1 mg/ml thiostrepton in 2% DMSO was added to induce tipA expression (+), and 12 μl of 2% DMSO was added to the control cultures (−). The plates were incubated at 30°C for 4 days. Induced expression of the S. coelicolor relA gene and the full-length and 1.6-kb 5′ region of At-RSH1 results in precocious antibiotic production (the blue pigment, actinorhodin) and precocious aerial hyphae formation, giving a white appearance to the mycelium.