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

SIMPLE SUMMARY: Erwinia mallotivora is the causal agent of papaya dieback disease in Malaysia, and its pathogenicity is less appreciated, especially from the molecular perspective. Our previous investigations proved that the hrpL/rpoE gene was one of the significant differentially expressed genes (DEGs) during early infection of E. mallotivora in papaya, suggesting this particular gene is important for infection. In this study, an in-depth analysis was performed using bioinformatics software on hrpL from E. mallotivora (EmhrpL) and its encoded protein (EmHrpL) obtaining crucial information including the conserved function and sequence motif, protein structural similarity with related homologs, and the possibility of being inhibited by a cognate inhibitor. Moreover, knockout (insertional mutational on DNA sequence) of the hrpL gene had caused mutant E. mallotivora (ΔEmhrpL) to be avirulent in four-month-old papaya plants. Here, the conclusion was that EmHrpL is indeed a necessary factor in E. mallotivora pathogenicity, and the findings on the potential inhibitor of this protein are useful for future studies to formulate a papaya dieback disease management programme. 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 (σ(70)) 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.