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

Date Published: January 31, 2017

Publisher: Public Library of Science

Author(s): Le Li, Weiguo Miao, Wenbo Liu, Shujian Zhang, Leandro Peña.


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.

Partial Text

Harpins, encoded by hypersensitive response and pathogenicity (hrp) genes of Gram-negative plant-pathogenic bacteria, are glycine-rich, heat stable, cysteine-free, protease-sensitive, acidic proteins that share common characteristics [1]. They can induce hypersensitive response (HR) and systemic acquired resistance in non-host plants through exogenous application [2]. It was reported that several harpins expressed in transgenic plants including tobacco, rice, and Arabidopsis induced host responses such as enhanced growth and drought tolerance [3–7]. According to their general characteristics, harpins are categorized into four major groups based on their protein similarity and domain structures: HrpN, HrpZ1, HrpW1, and Hpa1 groups [1]. HpaXm is a newly found harpin-like protein, cloned and identified from cotton leaf blight bacteria, Xanthomonas citri subsp. malvacearum—a synonym of X. campestris pv. malvacearum (Smith 1901–1978). Based on its composition of amino acids and other characteristics, hpaXm was distinct from other harpins and was classified into a new group [1]. Previous works showed that hpaXm was capable of inducing HR in plants and enhancing resistance to tobacco mosaic virus (TMV) in tobacco when infiltrated into the leaf apoplast using purified protein [8].

Unlike other common harpins, HpaXm, is classified in another group in terms of its amino acid composition and some special characteristics [1], such as a putative signal peptide detected in the N-terminal leader peptide [20]. We generated different transgenic tobacco lines expressing the full-length and the N-terminal signal peptide-like segment-deleted mutant of hpaXm respectively. Transgenic plants expressing the hpaXm and hpaXmΔLP genes conferred disease resistance to selected pathogens.

The N-terminal leader peptide (1-MNSLNTQIGANSSFL-15) was not necessary for trans-gene expressed hpaXm to elicit HR on tobacco leaves, to induce micro-HR in transgenic tobacco and to enhance the resistance of transgenic tobacco to TMV. However, the leader peptide was critical for the expressed hpaXm to be transported outside the plasma membranes of transgenic tobacco leaf tissues. Additionally, the N-terminal leader peptide (1-MNSLNTQIGANSSFL-15) of hpaXm acted as a signal peptide in transgenic tobacco, helping hpaXm to associate to the cell wall.

The data sets supporting the results of this article are included within the article and its additional files. The microarray data supporting the results of this article are available in the NCBI’s Gene Expression Omnibus repository, and are accessible through GenBank accession numbers DQ643828 (, AF485783 (, and AJ223969 (




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