Characterisation and Mutagenesis Study of An Alternative Sigma Factor Gene (hrpL) from Erwinia mallotivora Reveal Its Central Role in Papaya Dieback Disease

Abstract

The alternative sigma (σ) factor E, RpoE or HrpL, has been reported to be involved in stress- and pathogenicity-related transcription initiation in Escherichia coli and many other Gram-negative bacteria, including Erwinia spp. and Pseudomonas spp. A previous study identified the hrpL/rpoE transcript as one of the significant differentially expressed genes (DEGs) during early E. mallotivora infection in papaya and those data serve as the basis of the current project. Here, the full coding DNA sequence (CDS) of hrpL from E. mallotivora (EmhrpL) was determined to be 549 bp long, and it encoded a 21.3 kDa HrpL protein that possessed two highly conserved sigma-70 (σ⁷⁰) motifs-σR2 and σR4. Nucleotide sequence alignment revealed the hrpL from E. mallotivora shared high sequence similarity to rpoE/hrpL from E. tracheiphila (83%), E. pyrifoliae (81%), and E. tasmaniensis (80%). Phylogenetics analysis indicated hrpL from E. mallotivora to be monophyletic with rpoEs/hrpLs from Pantoea vagans, E. herbicola, and E. tracheiphila. Structural analysis postulated that the E. mallotivora's alternative σ factor was non-transmembranic and was an extracytoplasmic function (ECF) protein-characteristics shared by other σ factors in different bacterial species. Notably, the protein-protein interaction (PPI) study through molecular docking suggested the σ factor could be possibly inhibited by an anti-σ. Finally, a knockout of hrpL in E. mallotivora (ΔEmhrpL) resulted in avirulence in four-month-old papaya plants. These findings have revealed that the hrpL is a necessary element in E. mallotivora pathogenicity and also predicted that the gene can be inhibited by an anti-σ.

Keywords: Malaysia; bacterial disease; papaya; transcriptome sequencing.

Figure 1

Sequences of Targetron^® unique primers and retargeted intron. (a) Unique primers were generated by TargeTron^® algorithm (www.sigmaaldrich.com/targetronaccess) and used to produce retargeted intron for gene reverse splicing; (b) Sequence of the retargeted intron (350 bp) containing HindIII (AAGCTT) and BsrGI (TGTACA) restriction sites generated by the three unique primers through 1-step assembly PCR.

Figure 2

Sequence analysis of Emhrpl open reading frame (ORF) and motif finding on its translated protein. (a) Nucleotide sequence of EmhrpL (MK205448) and its deduced amino acids. The nucleotide sequences for BsaBI and EcoRV restriction sites are as indicated and the two conserved σ factor motifs on the amino acids, region R2 (red letters) and region R4 (green letters), are underlined. (b) Motif search on GenomeNet detected only two σ regions (or motifs) on EmHrpL, indicating the σ protein belongs to Group IV factor while (c) NPS@ server identified an HTH motif at position 149 of the amino acid (letters in red) corresponding to σR4 of the EmHrpL. (* = termination of translation by the stop codon TGA).

Figure 2

Sequence analysis of Emhrpl open reading frame (ORF) and motif finding on its translated protein. (a) Nucleotide sequence of EmhrpL (MK205448) and its deduced amino acids. The nucleotide sequences for BsaBI and EcoRV restriction sites are as indicated and the two conserved σ factor motifs on the amino acids, region R2 (red letters) and region R4 (green letters), are underlined. (b) Motif search on GenomeNet detected only two σ regions (or motifs) on EmHrpL, indicating the σ protein belongs to Group IV factor while (c) NPS@ server identified an HTH motif at position 149 of the amino acid (letters in red) corresponding to σR4 of the EmHrpL. (* = termination of translation by the stop codon TGA).

Figure 3

Molecular phylogenetic analysis of hrpL from E. mallotivora (Accession no. MK205448) and hrpL/rpoE gene sequences from related taxa by Maximum Likelihood (ML) method. A total of 18 nucleotide sequences were obtained from NCBI to construct the phylogenetic tree of sigma factors, and Pseudomonas carotovorum ssp. carotovorum served as an outgroup. The evolutionary history was inferred by using the ML method based on the Tamura–Nei model. The tree with the highest log likelihood (−2872.15) is shown.

Figure 4

Multiple sequence alignment of deduced amino acids of E. mallotivora hrpL (MK205448), Pantoea vagans rpoE (CP014128.2), E. tracheiphila rpoE (CP013970.1), E. pyrifoliae hrpL (AY532654.1), E. tasmaniensis rpoE (CU468135.1), and E. amylovora hrpL (U36244.1). Boxed are the extremely conserved sequences located on σR2 regions from six different bacterium species.

Figure 5

Predicted 3D structure of E. mallotivora’s σ factor (100% confidence) based on homology modelling of E. coli sigma factor E (RpoE) crystal structure. The region 4 (σR4) structure possesses the helix-turn-helix (HTH) motif as visualised.

Figure 6

Protein–protein interaction (PPI) study of molecular docking using ZDOCK protein-docking server. Structure as with visible surface at 40% transparency and secondary structures edited in PyMOL. (a) E.coli RNA polymerase sigma-E factor chain A (green) forming a complex with anti-σ factor RseA chain C (orange), and (b) EmHrpL (red) bound by anti-σ factor RseA chain C (orange) with a visible gap between the two proteins (white arrow). (c) EmHrpL without the anti-σ factor RseA viewed using PMV with a visible surface at 0% transparency.

Figure 7

Antibiotic selection of transformed E. mallotivora cells and confirmation of hrpL disruption in the genome by PCR. (a) Selection of putative mutants was carried out on LB agar supplemented with kanamycin (50 μg/mL). (b) For PCR validation, hrpL gene-specific primers were used to validate the putative mutants obtained from the selection step. The mutated strain (ΔEmhrpL) has a larger gene size due to insertion by the intron (lane 3) compared to non-mutant/wild type (lane 2). Lane 1 served as negative control.