Overexpression of Pti4, Pti5, and Pti6 in tomato promote plant defense and fruit ripening

Highlights

• Pti4/5/6-overexpressed tomato confer enhanced resistance to Pst DC3000.

• Transgenicis showed accelerated fruit ripening.

• Overexpression of Pti4/5/6 performed no obvious adverse impact on fruit traits.

• Pti4/5/6-overexpressed fruits exhibited increased activities of defense enzymes.

• Pti4, Pti5 and Pti6 degraded by 26S proteasome.

Abstract

Pseudomonas syringae pv. tomato (Pst) is a pathogenic microorganism that causes bacterial speck disease and affects tomato yield and quality. Pto is a disease resistant gene for plant to recognize and defense against Pst. Pto interacts with Pti (Pto interacting) proteins, which include three transcription factors, Pti4, Pti5, Pti6, and they were thought to be downstream of Pto-mediated pathway to promote the expression of disease-related genes. In the present work, the overexpression plants of Pti4, Pti5 or Pti6 were obtained by Agrobacterium-mediated transformation in tomato. The Pti4/5/6-overexpressed lines indicated enhanced expression of pathogenesis-related genes and resistance to pathogenic bacteria Pst DC3000. Meanwhile, the transgenic plants showed that Pti4/5/6 function in ripening but performed no obvious adverse influence on flowering time, seed-setting rate, weight and soluble solids content of fruits. Furthermore, Pti-overexpressed fruits exhibited increased enzymatic activities of phenylalnine ammonialyase, catalase, peroxidase and decreased content of malondialdehyde. Additionally, cell-free and in vivo ubiquitination assay indicated that Pti4, Pti5 and Pti6 degraded by 26S proteasome which suggested that these Pti transcription regulators’ functions could be regulated by ubiquitin-mediated post translational regulation in tomato.

Introduction

Tomato (Solanum lycopresicum) is one of the most important vegetable crops worldwide to be integrated as part of the human diet. However, there are a number of diseases that affect its yield and fruit quality, one of which is tomato bacterial speck disease caused by pathogenic microorganism Pseudomonas syringae pv. tomato (Pst) and brings about economic losses around the world [[1], [2], [3]]. Normally, the outbreaks of tomato speck disease occur when the pathogenic bacteria Pst colonized at internal tissues through natural openings and wounds on the plant surface and thus symbolized by a large amount of small brown necrotic spots (specks) on leaves and fruits [2,4]. In response to the pathogen attack, plants have evolved comprehensive strategies, especially disease resistance (R) gene-mediated defense mechanism [5]. In tomato, the disease resistance gene Pto (for P. s. pv. tomato), isolated by map-based cloning from resistant plant, functions in a gene-for-gene interaction with the avirulence (avr) avrPto/avrPtoB to perceive the existence of pathogen then confer resistance against Pst [[6], [7], [8], [9], [10], [11]].

To elucidate the Pto-mediated pathway, Pti (Pto interacting) proteins were screened by yeast two-hybrid and three interacted genes, named Pti4, Pti5 and Pti6 respectively, were identified. Pti4/5/6 encode proteins with characteristics of typical transcription factors and were thought to be the downstream components of Pto [12]. In addition, Pti4/5/6 showed similarity to tobacco ethylene-responsive element binding proteins [12,13], and indicated binding with the GCC-box cis element present in the promoter regions of ethylene response and pathogenesis-related (PR) genes [12,14]. Therefore, the identification of Pti4, Pti5 and Pti6 provides the linking between R gene and PR gene expression, Pto-mediated pathway and ethylene response pathway. When introduced into Arabidopsis thaliana, Pti4, Pti5, or Pti6 activated several PR genes’ expression [15]. Especially, overexpression of Pti4 increased the resistance of Arabidopsis plants to bacterial pathogen Pst DC3000 and fungal pathogen Erysiphe orontii [15]. In tomato plants, low abundance of Pti4 transcripts were found in young leaves, stems and green fruits, whereas high amounts were detected in old leaves, roots, and red fruits. Pti4 is induced when inoculated with Pst bacteria, and the abundance of Pti4 is also rapidly increased by exposure to salicylic acid, jasmonic acid, ethylene and wounding [14,16]. Pti4 protein could be phosphorylated by Pto kinase which enhanced its binding capacity to the cis element GCC-box [14]. Pti5 specifically induced by biotic stresses and Pti5-overexpression tomato accelerated pathogen-induced expression of GluB and Catalase [16,17]. Moreover, the recent study reported that Pti5 contributed to potato aphid (Macrosiphum euphorbiae) resistance in tomato [18]. The expression pattern of Pti6 in tomato tissues was similar to Pti4 except red fruits, in which they were less abundant, but Pti6 transcripts were not induced by pathogen inoculation in either RG-PtoR or RG-PtoS plants [14].

In our previous study, the interactions between Pto and Pti4/5/6 had been identified by co-immunoprecipitation in tobacco (Nicotiana benthamiana) leaf [19]. In the current work, the Pti4/5/6-overexpression tomato plants were separately generated to evaluate their effect on pathogen resistance and possible influence on fruit traits. The resulting Pti4/5/6-overexpressed lines showed enhanced resistance to Pseudomonas syringae pv. tomato and accelerated ripening of fruits but no obvious adverse effects on the other agronomic traits. The performance of Pti4/5/6 indicated their regulation in disease resistance and fruit ripening. Meanwhile, the degradation of Pti4, Pti5 and Pti6 detected by cell-free protein stability and in vivo ubiquitination suggested the ubiquitin-proteasome pathway involved in regulation of these Pto interacting proteins.