Jasmonic Acid and Ethylene Signaling Pathways Regu

Authors: Nurmi Pangesti Michael Reichelt Judith E van de Mortel Eleni Kapsomenou Jonathan Gershenzon Joop J A van Loon Marcel Dicke Ana Pineda

Publish Date: 2016/11/15

Volume: 42, Issue: 12, Pages: 1212-1225

PDF Link

Abstract

Beneficial soil microbes can promote plant growth and induce systemic resistance ISR in aboveground tissues against pathogens and herbivorous insects Despite the increasing interest in microbialISR against herbivores the underlying molecular and chemical mechanisms of this phenomenon remain elusive Using Arabidopsis thaliana and the rhizobacterium Pseudomonas simiae WCS417r formerly known as P fluorescens WCS417r we here evaluate the role of the JAregulated MYC2branch and the JA/ETregulated ORA59branch in modulating rhizobacteriaISR to Mamestra brassicae by combining gene transcriptional phytochemical and herbivore performance assays Our data show a consistent negative effect of rhizobacteriamediated ISR on the performance of M brassicae Functional JA and ETsignaling pathways are required for this effect as shown by investigating the knockout mutants dde22 and ein21 Additionally whereas herbivory mainly induces the MYC2branch rhizobacterial colonization alone or in combination with herbivore infestation induces the ORA59branch of the JA signaling pathway Rhizobacterial colonization enhances the synthesis of camalexin and aliphatic glucosinolates GLS compared to the control while it suppresses the herbivoreinduced levels of indole GLS These changes are associated with modulation of the JA/ETsignaling pathways Our data show that the colonization of plant roots by rhizobacteria modulates plantinsect interactions by prioritizing the JA/ETregulated ORA59branch over the JAregulated MYC2branch This study elucidates how microbial plant symbionts can modulate the plant immune system to mount an effective defense response against herbivorous plant attackersPlants as primary producers in terrestrial ecosystems are exposed to various attackers with insect herbivores among the most important ones To survive plants have evolved physical and chemical barriers as defenses against insect herbivores Upon recognition of insect elicitors plants use hormones that regulate signaling pathways to reprogram their transcriptome and metabolome thus strengthening their defense Bodenhausen and Reymond 2007 De Vos et al 2005 Reymond et al 2004 In Brassicaceous plants including Arabidopsis thaliana glucosinolates GLS are the main defensive compounds that confer plant resistance against insect herbivores Beekwilder et al 2008 Howe and Jander 2008 Mewis et al 2006 Müller et al 2010 The two most abundant classes of GLS are aliphatic and indolic GLS depending on whether the side chain is derived from the amino acid methionine or tryptophan respectively Halkier and Gershenzon 2006 Feeding by specialist and generalist leafchewing insects triggers enhanced synthesis of aliphatic and indolic GLS Kos et al 2012 Verhage et al 2011 More recent studies show that other compounds such as camalexin a brassicaceous indolic phytoalexin also contribute to plant resistance against herbivores Kettles et al 2013 Kusnierczyk et al 2008 Prince et al 2014 Schlaeppi et al 2008 Unraveling how plant signaling pathways and crosstalk between pathways regulate the synthesis of defensive compounds in the context of multitrophic interactions has only just begunThe signaling pathway regulated by the plant hormone jasmonic acid JA is the core pathway regulating resistance to leafchewing herbivores Erb et al 2012 Farmer and Ryan 1992 Howe and Jander 2008 Kessler and Baldwin 2002 through formation of physical barriers such as trichomes and enhanced synthesis of defensive compounds such as GLS Erb et al 2012 Howe and Jander 2008 The JAsignaling pathway has two branches that crosscommunicate with other hormonal pathways such as the ethylene ET and abscisic acid ABA pathways through the use of common transcription factors Kazan and Manners 2008 Lorenzo and Solano 2005 The transcription factor ORA59 is one of the main integrators of the JA and ETsignaling pathways Lorenzo et al 2003 Pre et al 2008 whereas transcription factor MYC2 is one of the main integrators of JA and ABAsignaling pathways Abe et al 2002 Vos et al 2013 Each of these two transcription factors activates different sets of JAresponsive genes Kazan and Manners 2013 Lorenzo and Solano 2005 Pieterse et al 2012 In A thaliana MYC2 regulates the biosynthesis of defensive compounds such as camalexin and GLS Kazan and Manners 2013 Schweizer et al 2013 In line with this feeding by the leafchewing insects Pieris rapae and Helicoverpa armigera induces the MYC2branch and enhances the expression of the JAresponsive gene Vegetative Storage Protein 2 VSP2 Verhage et al 2011 The MYC2branch also has an important function in mediating the ability of beneficial microbes to prime plant defenses to pathogens Pozo et al 2008 An intriguing question then is whether the MYC2branch also plays a role in the ability of beneficial microbes to increase plant resistance to insect herbivoresPlants host a diversity of microbes including beneficial microbes in the rhizosphere that can affect plant defense and growth The influence of beneficial microbes on plant defense and growth is mediated by their effect on plant signaling pathways that not only regulate microbial colonization but also trigger significant changes in plant gene expression metabolism and physiology Cartieaux et al 2008 Van de Mortel et al 2012 Verhagen et al 2004 Weston et al 2012 Interestingly several species of rootassociated microbes from the genera Pseudomonas Bacillus and Trichoderma enhance plant immunity through a mechanism called induced systemic resistance ISR known to inhibit growth and development of various insect herbivores and pathogens Pangesti et al 2015 Pineda et al 2010 Song et al 2013 ValenzuelaSoto et al 2010 Intact JA and ET hormonal signaling pathways are required to induce ISR by several rootassociated microbes such as P fluorescens WCS417r against pathogens Pieterse et al 1998 Based on the whole genome sequence comparison this rhizobacterium isolate recently has been renamed into Pseudomonas simiae WCS417r Berendsen et al 2015 However it is unknown if intact JA and ET signaling pathways also regulate ISR against insect herbivores Furthermore it remains to be elucidated how plants regulate chemical defense against insect herbivores upon colonization by rootassociated beneficial microbesThe present study investigates how colonization by the rhizobacterium P simiae WCS417r affects plant defense against the leafchewing insect Mamestra brassicae Previous studies have found that this rhizobacterium triggers an enhanced expression of the JAregulated gene LOX2 and the JA/ETregulated genes PDF12 and HEL upon feeding by the generalist caterpillars M brassicae and Spodoptera exigua Pangesti et al 2015 Van Oosten et al 2008 However whether the JAregulated MYC2branch or the JA/ETregulated ORA59branch is modulating plant defense in rhizobacteriamediated ISR against insects is unknown To investigate this gene transcription plant chemistry and performance of the herbivore M brassicae were analyzed in vitro in wild type A thaliana Col0 and in mutants defective in the JA pathway ie dde22 and myc2 in the ET pathway ie ein21 and in the JA/ET pathway ie ora59 We hypothesized that rhizobacteriatreatment of plants 1 triggers enhanced expression of the JA/ETregulated genes ORA59 and PDF12 and of the JAregulated genes MYC2 and VSP2 upon feeding by M brassicae 2 increases the synthesis of glucosinolates and camalexin upon feeding by M brassicae and 3 results in stronger resistance to M brassicae via the JA and ETsignaling pathwaysThe rifampicinresistant nonpathogenic epiphyte rhizobacterium strain P simiae WCS417r abbreviated as Ps WCS417r was used Rhizobacteria were grown on King’s B KB medium agar plates containing rifampicin 25 μg ml−1 for 48 h at 28°C Pieterse et al 1996 Prior to inoculation on plant roots a single colony of the strain was transferred to KB liquid medium amended with rifampicin as indicated above and was grown in an incubator shaker for 24 h at 200 rotations per minute rpm at 25°C The bacterial cells were collected resuspended in 10 mM MgSO4 and washed three times with 10 mM MgSO4 Afterwards the bacterial cells were resuspended in 10 mM MgSO4 and adjusted to a cell density of 1x109 colony forming units cfu ml−1 OD660 = 10Colonization of A thaliana roots by Ps WCS417r was quantified in wild type plants and mutants to confirm that the colonization met the required threshold for ISR of 105 cfug−1 root Raaijmakers et al 1995 The rhizobacteria quantification was done following the method described in Pangesti et al 2015 with slight modification Roots were harvested weighed and shaken vigorously for 1 min in 10 ml of 10 mM MgSO4 containing 05 g of glass beads 425–600 μm SigmaAldrich Proper dilutions were plated onto KB agar medium supplemented with 25 μg ml−1 rifampicin to select for rifampicinresistant fluorescent Pseudomonas spp Pieterse et al 1998 The dilution plates were incubated for 48 h at 28°C after which the number of cfu per mg root fresh weight was determined

Keywords: