Structural basis of a novel repressor, SghR, controlling Agrobacterium infection by cross-talking to plants

Abstract

Agrobacterium tumefaciens infects various plants and causes crown gall diseases involving temporal expression of virulence factors. SghA is a newly identified virulence factor enzymatically releasing salicylic acid from its glucoside conjugate and controlling plant tumor development. Here, we report the structural basis of SghR, a LacI-type transcription factor highly conserved in Rhizobiaceae family, regulating the expression of SghA and involved in tumorigenesis. We identified and characterized the binding site of SghR on the promoter region of sghA and then determined the crystal structures of apo-SghR, SghR complexed with its operator DNA, and ligand sucrose, respectively. These results provide detailed insights into how SghR recognizes its cognate DNA and shed a mechanistic light on how sucrose attenuates the affinity of SghR with DNA to modulate the expression of SghA. Given the important role of SghR in mediating the signaling cross-talk during Agrobacterium infection, our results pave the way for structure-based inducer analog design, which has potential applications for agricultural industry.

Keywords: LacI family; SghR; crystal structure; gene regulation; host–pathogen interaction; inhibition mechanism; microbial pathogenesis; salicylic acid; sucrose.

Figure 1.

SghR-SghA involved in plant tumorigenesis and SghR regulating SghA via binding to its promoter region. A, tumorigenicity assay for A. tumefaciens A6 and its derivatives using carrot discs. Representative images (bottom) were taken and fresh weight of tumors (top) were recorded at 6 weeks after inoculation. ΔsghR, ΔsghA, and ΔsghRA indicate the in-frame deletions of sghR, sghA, and both genes, respectively. The error bars represent SD from 6 repeats. A nonparametric 1-way analysis of variance was performed in GraphPad for statistical analyses. **, p < 0.001; ***, p < 0.0001. B, SghR represses the transcription of the gene indicated by RT-PCR. The transcription level of 16S RNA in two strains is also presented. C, identification of SghR-binding site in sghA promoter region using Dye Primer sequencing on an automated capillary DNA analyzer. The double-ended arrow shows the region in the sghA promoter protected by SghR during DNase I digestion. The sghA promoter region and DNase I are incubated either in the presence (+SghR) or absence (−SghR) of SghR protein. Electropherograms of ddNTP (ddATP, ddTTP, ddCTP, and ddGTP) panels displayed in the figure are the sequencing results using forward primer (6-FAM labeling). D, depiction of the SghR-binding site in the promoter region of sghA. The binding site is shown in red letters. The two opposite red arrows indicate the partial palindrome feature presented in the binding site. −35 and −10 regions of sghA were predicted by the online program BPROM, and sequences were labeled with blue lines. The pentacle indicates the transcription start site, predicted by an online server (http://www.fruitfly.org/seq_tools/promoter.html). The translation start site of SghA is also indicated by a blue arrow. E, in vitro gel shift assay of SghR with the identified binding site. Various ratios of protein to DNA were used. Left, native-PAGE gel stained by DNA-staining dye. Right, the same gel stained by Coomassie brilliant blue (CBB). D and Pro represent DNA and protein, respectively. Pro:D = 1:1, 1:2, and 2:1 indicate that the molar ratios of protein dimer to dsDNA are 1 to 1, 1 to 2, and 2 to 1, respectively. This is native PAGE; no protein marker was included in the gel, as the charge of protein also contributes to its migration rate in addition to its molecular weight.

Figure 2.

Binding affinity of SghR with Ppa0305 promoter DNA (A), dsDNA from sghA promoter (B), sucrose (C), and sucrose-binding SghR with dsDNA (D). Panel A is a control experiment for panel B. B and D, ITC data of titrating dsDNA into SghR and SghR–sucrose mixture, respectively. Raw data are shown in the upper panels, whereas the fitted curves are shown in the lower panels. Both profiles were fitted into one set of the site model. C, quantification of binding affinity between SghR and sucrose by microscale thermophoresis. Data points indicate the difference in normalized fluorescence (‰) generated by SghR protein, and the curve indicates the calculated fit.

Figure 3.

Crystal structure of the apo SghR and its dimerization. A, cartoon representation of the overall structure of SghR monomer. The final model (chain A, colored cyan) comprises 275 of 350 residues, representing the C-terminal core domain. Because the electron density for the N-terminal region forming the first four helices observed in the DNA-bound structure was not observed, the labeling of the secondary structure elements starts from α5. Each SghR monomer is composed of two subdomains. B, cartoon representation of the structure of apo SghR dimer. Chain A and chain B are colored cyan and light magenta, respectively. (The primes refer to the second monomer within a dimer, same as below). The dimerization interface is framed by solid and dashed black boxes, respectively. C, enlarged view of the dimer interface involving β2 and α5 in the N subdomains. The residues involved in dimerization are labeled and shown as sticks, and their electron densities (2Fo-Fc map contours to 1σ) were shown as a blue mesh. The hydrogen bonds formed between L112-V114′, L112′-V114, E88-S95′, and E88-S95′ are shown as dashed lines. D, enlarged view of the dimer interface involving α11 and the loop region between β9 and β10 in the C subdomains. Residues involved in dimerization are shown as sticks, and their electron densities (2Fo-Fc map contours to 1σ) are shown as blue mesh.

Figure 4.

Crystal structure of SghR in complex with its operator DNA. A, cartoon representation of the structure of SghR-DNA in two views. The chains A and B of SghR protein are colored cyan and light magenta, whereas the dsDNA duplex is colored gold (a) and light green (b), respectively (same as below). The first four N-terminal helices are labeled. B, comparison of the natural operator DNA sequence with the modified symmetric DNA sequence. The upper panel shows the natural operator DNA sequence determined by the DNase I footprinting assay, which is 24 bp long, containing the pseudopalindromic region indicated by the blue inverted arrows. The lower panel shows the symmetric DNA sequence used for the crystallization of the SghR-DNA complex, which is a symmetric (16 bp long) DNA with a two-nucleotide 5′ overhang on each strand. The palindrome region is also indicated by the inverted blue arrows, and the nucleotides that were mutated to generate a perfect palindrome are colored blue. C, structural illustration of residues on the N-terminal DNA-binding domain of monomer A interacting with one-half of the DNA duplex. The interacting residues are labeled and shown in stick model with nitrogen and oxygen atoms displayed in blue and red, respectively. The electron densities (Fo-Fc omit map contours to 3σ) of these residues are shown as a blue mesh as well. D, illustration of the interactions between SghR and the DNA duplex. The DNA bases are labeled (guanine = G; adenine = A; cytosine = C; thymine = T), and the pentose sugars are numbered from the 5′ to 3′ end. The black dotted lines represent hydrogen bonds between the respective SghR residues and DNA phosphate backbone or DNA bases. The residues of SghR involved in the interactions are colored cyan for chain A and light magenta for chain B. The black dot in the middle of nucleotides 10 and 11 represents the 2-fold symmetry of the DNA in the crystal structure.

Figure 5.

Crystal structure of SghR in complex with sucrose and inducer binding pocket comparison between LacI and SghR. A, cartoon representation of the structure of SghR–sucrose complex. SghR is colored light orange. Sucrose is shown as a stick, with carbon and oxygen atoms displayed as cyan and red, respectively. The color codes are the same below. B, unbiased difference Fourier electron density map (Fo-Fc) of sucrose (Fo-Fc map contours to 3σ). The interacting residue E310 of SghR and one water molecule are shown as well. C, detailed interaction of SghR with sucrose. The residues involved in sucrose binding are shown as sticks. The water molecules involved in the hydrogen-bonding network are shown as pink spheres. The residues involved in sucrose binding are labeled and shown as sticks, with electron densities (2Fo-Fc map contours to 1σ) indicated as blue mesh. The hydrogen bonds formed between residues/water molecules with sucrose are shown as dashed lines. D, detailed interactions of LacI with IPTG (PDB code 2P9H). For comparison, the residues involved in interacting with IPTG were shown as sticks. The hydrogen bonds between IPTG and LacI or the water-mediated hydrogen bond are shown as dash lines.

Figure 6.

Structural comparison of SghR-sucrose and SghR-DNA complexes. A, superposition of the individual N-subdomains based on the alignment matrix of individual C-subdomains from both SghR–sucrose and SghR–DNA complexes. The N-subdomains of SghR-sucrose are colored light blue, whereas the N-subdomains of SghR-DNA complex are colored cyan for chain A and light magenta for chain B. The dashed-lined boxes demonstrate the regions (residues 145–174 and 321–337, as indicated by the double-ended arrows in panel B) exhibiting relatively large conformational changes. The color codes for chain A and chain B are the same below. B, the Cα deviation for each residue on the N-subdomains based on the alignment methods in panel A for SghR–sucrose and SghR–DNA complexes. The two double-ended arrows indicate the relatively large conformational change regions between the two complex structures. C and D, conformational changes of the key residues involved in domain interactions based on the comparison of SghR-DNA complex with SghR-sucrose complex. C, Arg19 (chain A) and Thr133′ (chain B) from SghR-DNA complex form a hydrogen bond (shown as dashed line), whereas in SghR-sucrose complex, Thr133′ (colored gray) shows a different conformation. D, His111 (chain A) and Tyr129′ (chain B) form a hydrogen bond (indicated by a dashed lines), whereas this hydrogen bond is abolished in the SghR-sucrose complex because of the conformational change of both His111 and Tyr129′ (colored gray). In addition, in the SghR-DNA complex, Arg67 and His111 establish a π–cation interaction (shown as a double-ended arrow), whereas because of the conformation change of His111, this interaction is also likely to be abolished in the SghR–sucrose structure. In panels C and D, their electron densities (2Fo-Fc map contours to 1σ) are shown.