Flavescence Dorée Phytoplasma Has Multiple ftsH Genes that Are Differentially Expressed in Plants and Insects

Abstract

Flavescence dorée (FD) is a severe epidemic disease of grapevines caused by FD phytoplasma (FDP) transmitted by the leafhopper vector Scaphoideus titanus. The recent sequencing of the 647-kbp FDP genome highlighted an unusual number of genes encoding ATP-dependent zinc proteases FtsH, which have been linked to variations in the virulence of "Candidatus Phytoplasma mali" strains. The aims of the present study were to predict the FtsH repertoire of FDP, to predict the functional domains and topologies of the encoded proteins in the phytoplasma membrane and to measure the expression profiles in different hosts. Eight complete ftsH genes have been identified in the FDP genome. In addition to ftsH6, which appeared to be the original bacterial ortholog, the other seven gene copies were clustered on a common distinct phylogenetic branch, suggesting intra-genome duplication of ftsH. The expression of these proteins, quantified in plants and insect vectors in natural and experimental pathosystems, appeared to be modulated in a host-dependent manner. Two of the eight FtsH C-tails were predicted by Phobius software to be extracellular and, therefore, in direct contact with the host cellular content. As phytoplasmas cannot synthesize amino acids, our data raised questions regarding the involvement of FtsH in the adaptation to hosts via potentially enhanced recycling of phytoplasma cellular proteins and host protein degradation.

Keywords: membrane proteases; phytoplasmas; protein metabolism; virulence.

Figure 1

FtsH proteins deduced from the eight ftsH genes of the genome of the FDP strain FD92 with the different functional domains characteristic of FtsH: two predicted transmembrane domains (TM1 and TM2), ATP-binding sites walker A and walker B (wA and wB), pore signature (PS), ATP-hydrolysis site (SRH), the HExxH zinc-binding motif (Zn), the third Zn-ligand-binding glutamic acid residue (E), the third coordinating aspartic acid residue important for the proteolytic activity (D) and a leucine-zipper residue for substrate binding (Leu-zip). The E. coli and B. subtilis FtsH proteins are shown as references.

Figure 2

Molecular phylogenetic analysis by the maximum likelihood method based on the proteic sequences [32]. The analysis involved 42 amino acid sequences. Evolutionary analyses were conducted in MEGA6 [33]. The 8 FtsH from FDP are in bold. The tree is drawn to scale, with branch lentghs measured in the number of substitutions per site.

Figure 3

(A) Position of ftsH genes on the FDp genome (strain FD92). Black arrows indicate CDS on the (+) strand, and white arrows indicate CDS on the (−) strand. (B) Genomic context of ftsH genes. CDS on the (+) strand are represented with a right arrow, and those on the (−) strand are represented with a left arrow. Black arrows correspond to the FtsH CDS, hatched boxes to domains with similarity to ABC transporters substrate-binding proteins, and gray arrows to CDS associated with a putative transporter function.

Figure 4

Relative ftsH gene expression (log mean normalized expression (MNE)) of FDP strain FD92 in plant hosts (CS: grapevine and broad bean) and in insect vectors (Ev: E. variegatus and St: S. titanus). Different letters refer to different mean values calculated for each host (Wilcoxon test, p < 0.05).

Figure 5

Relative gene expression (RGE) of ftsH genes in infected grapevine and leafhopper vector S. titanus (left) and in infected broad bean and leafhopper vector E. variegatus (right). Different letters refer to different mean values (Wilcoxon test, p < 0.05).

Figure 6

Schematic representation of possible insertion of FtsH in the FD phytoplasma membrane. FtsH are represented as hexamers anchored in the phytoplasma membrane. The C-tail is facing the inside of the phytoplasma (left) or the outside (right). When facing the inside, FtsH can degrade protein into peptides. When facing the outside, FtsH can degrade host’s proteins and peptides can either stay in the outside of the phytoplasma, or can be imported in the phytoplasma via an unknown way.