The signal peptide-like segment of hpaXm is required for its association to the cell wall in transgenic tobacco plants

Abstract

Harpins, encoded by hrp (hypersensitive response and pathogenicity) genes of Gram-negative plant pathogens, are elicitors of hypersensitive response (HR). HpaXm is a novel harpin-like protein described from cotton leaf blight bacteria, Xanthomonas citri subsp. malvacearum—a synonym of X. campestris pv. malvacearum (Smith 1901–1978). A putative signal peptide (1-MNSLNTQIGANSSFL-15) of hpaXm was predicted in the nitroxyl-terminal (N-terminal)by SignalP (SignalP 3.0 server). Here, we explored the function of the N-terminal leader peptide like segment of hpaXm using transgenic tobacco (Nicotiana tabacum cv. Xanthi nc.). Transgenic tobacco lines expressing the full-length hpaXm and the signal peptide-like segment-deleted mutant hpaXmΔLP were developed using transformation mediated by Agrobacterium tumefaciens. The target genes were confirmed integrated into the tobacco genomes and expressed normally. Using immune colloidal-gold detection technique, hpaXm protein was found to be transferred to the cytoplasm, the cell membrane, and organelles such as chloroplasts, mitochondria, and nucleus, as well as the cell wall. However, the deletion mutant hpaXmΔLP expressed in transgenic tobacco was found unable to cross the membrane to reach the cell wall. Additionally, soluble proteins extracted from plants transformed with hpaXm and hpaXmΔLP were bio-active. Defensive micro-HR induced by the transgene expression of hpaXm and hpaXmΔLP were observed on transgenic tobacco leaves. Disease resistance bioassays to tobacco mosaic virus (TMV) showed that tobacco plants transformed with hpaXm and with hpaXmΔLP exhibited enhanced resistance to TMV. In summary, the N-terminal signal peptide-like segment (1–45 bp) in hpaXm sequence is not necessary for transgene expression, bioactivity of hpaXm and resistance to TMV in transgenic tobacco, but is required for the protein to be translocated to the cell wall.

WY195, a new inducible promoter from the rubber powdery mildew pathogen, can be used as an excellent tool for genetic engineering

“…and drought-inducible promoter. We have confirmed that WY195 can regulate the Similarly, hrp gene transgenic plants showed resistance to plants [23,64,65]. Results in our study supported the view, hpaXm also induced resistance when acting both intercellular and intracellular.…”

“…Soluble protein extracted from hpaXm transgenic tobacco can stimulate a HR in tobacco. Endogenous expression of hpaXm can cause a micro-HR in tobacco, and can induce tobacco resistance to TMV [23].…”

Functional regions of HpaXm as elicitors with specific heat tolerance induce the hypersensitive response or plant growth promotion in nonhost plants

“…The former subgroup contains a threonine residue, but the latter subgroup contains a cysteine in the corresponding position [ 14 ]. Moreover, a putative signal peptide (1–15 aa) of HpaXm was predicted in the N-terminal by SignalP (SignalP 3.0 server), which is required for HpaXm to be translocated to the cell wall [ 27 ]. A putative signal peptide (1–15 aa) was also found in HpaG and HpaXac, but was not found in Hpa1Xoo and Hpa1Xoc.…”

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“…When applied exogenously, hpaXm induces plants to produce a HR and enhances the resistance of tobacco to Tobacco mosaic virus (TMV). Li et al (2017) transformed hpaXm and its signal peptide fragment HpaXm LP into N Nicotiana tabacum. Soluble protein extracted from hpaXm transgenic tobacco can stimulate a HR in tobacco.…”

“…Soluble protein extracted from hpaXm transgenic tobacco can stimulate a HR in tobacco. Endogenous expression of hpaXm can cause a micro-HR in tobacco, and can induce tobacco resistance to TMV (Li et al, 2017).…”