Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci Biotic factors that induce the tomato Ve1 R-gene Christian Danve Castroverde, Xin Xu, Ross N. Nazar, Jane Robb ⁎ Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada ARTICLE INFO Keywords: Tomato Verticillium wilt Ve R-locus Gene expression Ave1 Hormone regulation ABSTRACT In tomato, Verticillium resistance is determined by the Ve gene locus encoding two leucine-rich repeat-receptor- like proteins (Ve1, Ve2). The resistance function usually is attributed to Ve1 alone, with two known alleles: Ve1, encoding a resistance protein, and ve1, with a premature stop codon encoding a truncated product. We have examined further Ve-gene expression in resistant and susceptible near-isolines of Verticillium-infected Craigella tomatoes, using both quantitative RT-PCR and an alternative RFLP assay. Ve1 is induced differentially in re- sistant and susceptible plants, while Ve2 is constitutively expressed throughout disease development. Contrary to their putative role in Verticillium resistance, these profiles were observed even with compatible Verticillium in- teractions, some bacterial pathogens, and transgenic tomato plants expressing the fungal Ave1 effector. This suggests broader roles in disease and/or stress. To determine the contribution of plant hormones, abscisic acid, methyl jasmonate, naphthaleneacetic acid or salicylic acid were infused independently via the tomato root and effects on Ve1 induction were confirmed using biosynthesis mutants. While all the hormones modulated Ve1- gene induction, abscisic acid and salicylic acid were not required while jasmonic acid appears to play a more direct role. 1. Introduction In tomato the Ve-gene locus governs resistance to race1 isolates of Verticillium dahliae (Vd1) and V. albo-atrum (Vaa1). The Ve-locus com- prises two genes, Ve1 and Ve2, which lie head-to-head with a short intergenic region between [1,2]. Both genes encode putative trans- membrane proteins that appear to belong to the RLP class of plant re- sistance proteins [3]. Although somewhat controversial [1,2], the re- sistance function generally is attributed to Ve1 alone, this gene having two known alleles; one is dominant (Ve1) and encodes a full length R- protein (117 kD) while the other, is recessive (ve1) and has a premature stop codon resulting in a severely truncated product [2]. Most molecular studies to date have been directed at characterizing the gene family [1,2] and the functional domains of the expressed Ve1 and Ve2 proteins [4,5] as well as studies on the possible fungal effector molecule, termed Ave1 [6]. Some elements of the putative associated signalling pathways also have been proposed, although much of this work has been carried out in hosts other than tomato [2,7–9]. Despite being important for a basic understanding of function, however, little is known about the actual expression of Ve1 and Ve2. The present paper focuses on the induction and expression of the two Ve genes. To clarify previous inconsistencies [2,10], we have re- examined the expression of Ve1 and Ve2 in tomatoes infected by Vd1 in greater detail using compatible and incompatible interactions and two different assay strategies. In addition, to investigate the mechanism of Ve1-gene induction we have studied induction by other wilt and bac- terial pathogens, by the putative Ave1 effector and by specific plant hormones linked to plant defense and stress. 2. Materials and methods 2.1. Plant materials and growth The plants used in these studies are summarized in Table 1. They include two near-isolines of tomato (Solanum lycopersicum L.) cv Crai- gella with (CR) or without (CS) the Ve1 allele that confers resistance to V. dahliae, race 1 or V. albo-atrum, race 1. Comparisons also are made with cv Castlesmart (CST), cv Rheinland Ruhm (RR), cv VF 36 (VF) and cv Heinz 5108 (H). Seeds were surface sterilized in a 1% hypochlorite solution for 10 min, then rinsed in distilled water three times (total rinse time = 1 h) and planted in 17 × 13 × 6 cm 3 Kord cell flats (i.e. 6 plants/flat) containing a 3:2:1 mixture of Promix BX (Premier Horti- culture Lteé, Rivie‘re-du-Loup, Que., Canada), vermiculite and turface mvp (Profile Products LLC, Buffalo Grove, Illinois, USA). Plants were maintained in Percival growth chambers (Percival Scientific Inc., Perry, Iowa) with a cycle of 14 h of light (2.5 × 10 3 μ Einsteins m -2 s -1 ) and 10 h of dark and fertilized weekly with Hoagland’s solution [11]. After about 1 week the seedlings were transplanted again to larger pots http://dx.doi.org/10.1016/j.plantsci.2017.09.015 Received 2 June 2017; Received in revised form 11 September 2017; Accepted 20 September 2017 ⁎ Corresponding author. E-mail address: jrobb@uoguelph.ca (J. Robb). Plant Science 265 (2017) 61–69 Available online 28 September 2017 0168-9452/ © 2017 Elsevier B.V. All rights reserved. MARK