Defense Responses Of Tomato Plants Mediated By Helicoverpa Zea Gut-associated Bacteria And Their Relative Mechanisms

Abstract:Herbivorous insects harbor many microbes in their guts.These gut-associated microbes not only possess important genetic information of their hosts,but also mediate the host behavior and physiology.Insect symbionts-modulated plant defenses have been observed in beetles and piercing-sucking insects,but the role of caterpillar-associated bacteria in regulating plant-induced defenses has not been adequately examined.We identified bacteria from the regurgitant of field-collected corn earworm Helicoverpa zea larvae using 16S rRNA gene sequencing and MALDI-TOF mass spectrometry.All isolated bacteria were screened through inoculating lab-reared H.zea on tomato plants(Solanum lycopersicum).The GOX activities in the labial glands of caterpillars and PPO activities in tomato leaves were analyzed.A combination of biochemical,molecular,and confocal-electron microscopy methods was used to determine the role of caterpillar-associated bacteria in mediating defense responses in tomatoes.Infestation by H.zea caterpillars inoculated with E.ludwigii led to different responses in tomato plants in defense signaling pathways,defense-related enzyme activities and phytohormone contents relative to tomatoes infested by E.ludwigii uninoculated caterpillars.By the technique of SDS-PAGE and Western blot,we were able to compare the difference in the content of GOX on tomato leaves that secreted by E.ludwigii-inoculated and uninoculated caterpillars.Additionally,a pair of specific primers were designed to examine the existence of E.ludwigii on tomato leaves damaged by H.zea.The aim of this study was to reveal the mechanism how H.zea gut-associated bacteria mediate induced defenses in tomato plants.The main results were as follows:1.Identification of gut-associated bacteria isolated from field and lab-grown H.zeaThe regurgitant collected from both field and lab-grown caterpillars was cultured by the traditional culturing method.The amount of bacteria in the regurgitant of caterpillars collected from field colonies was much higher that in the regurgitant of lab-reared caterpillars.In total,sixteen bacterial isolates were randomly selected and amplified by universal primers of 16S rRNA gene.The PCR products of selected isolates were sequenced,and sequencings were blasted in NCBI database and RDP database.Moreover,the Biotyper technique was used to double-check the identification of all isolates.Based on these methods,fourteen genuses of bacteria were identified.The isolated bacteria were identified as members of the genera Enterobacter(5 isolates),Erwinia(1 isolate),Klebsiella(1 isolate),Pantoea(3 isolates),Rahella(2 isolates),Raoultella(1 isolate),and Serratia(1 isolate).However,eight bacteria isolated from lab-reared caterpillars belonged to the same genus,Enterococcus,with high similarity.2.Screening for bacteria that mediated immune responses of both insects and plantsThe GOX activies of homogenized labial glands were found much higher in field-collected H.zea than those in lab-reared colonies.By inoculating all of 14 bacteria isolated from field-grown caterpillars to lab-reared caterpillars,four bacteria increased GOX activities,and they were identified as 3-1,(Rahnella aquatilis),2b(E.ludwigii),5-1(Enterobacter sp.)and 6-1(Pantoea agglomerans),respectively.Similarly,all of the isolated bacteria were inoculated to tomato plants,three isolates were found to induce PPO activities in tomato leaves,and they were 3-1(Rahnella aquatilis),2b(E.ludwigii)and 5-1(Enterobacter sp.).3.E.ludwigii-inoculated caterpillars induce JA-responsive defenses in tomato plantsTomato plants damaged by buffer-inoculated H.zea larvae(control)had lower PPO activities than those in plants damaged by E.ludwigii-inoculated larvae.The weight of larvae fed on detached tomato leaves damaged by E.ludwigii-inoculated H.zea larvae was significantly lower than that of larvae fed on tomato leaves damaged by buffer-inoculated larvae.Moreover,tomato plants damaged by E.ludwigii-inoculated H.zea larvae had significantly higher expression levels of the JA-responsive Pin2,CysPI and PPOF compared to plants damaged by buffer-inoculated caterpillars,while the expression level of SA-responsive Pr1a(P4)was lower in tomato plants damaged by E.ludwigii-inoculated larvae.4.The suppression of E.ludwigii was dose-dependent when applied to wounded plantsWhen tomato plants were inoculated with different concentration of E.ludwigii,only10~9 CFU/mL of E.ludwigii was found to suppress PPO activities in tomato leaves.In addition,E.ludwigii inoculation led to decrease in JA-responsive Pin2 transcripts and increase in SA-related Pr1a(P4)transcripts relative to un-inoculated control.5.The inoculation of E.ludwigii affected H.zea salivary secretionTo determine if the inoculation of E.ludwigii affects secretion of GOX from the saliva of H.zea larvae,GOX proteins were detected on wounded sites of tomato leaves using a specific GOX antibody.Western blot analysis showed that E.ludwigii-inoculated H.zea secreted much more GOX protein onto tomato leaves than that secreted by buffer-inoculated larvae.6.Saliva from E.ludwigii-inoculated caterpillar triggers tomato JA-regulated defensesAblation of the spinneret significantly reduced PPO activities compared with plants damaged by E.ludwigii-inoculated larvae with intact spinnerets.For buffer-inoculated larvae,there was an insignificant decline of PPO activities in tomato plants damaged by buffer-inoculated larvae after the spinnerets were ablated.There were no significant differences in PPO activities of tomato plants damaged by only buffer or E.ludwigii-inoculated larvae when the spinnerets of all treated H.zea larvae were ablated.In addition,higher PPO activity was observed in tomato plants treated with saliva collected from E.ludwigii-inoculated larvae than that in plants treated with saliva collected from buffer-inoculated caterpillars.Moreover,salivary gland homogenates of E.ludwigii-inoculated H.zea larvae were found to elicit higher PPO activities in tomato plants as compared to those treated with the gland homogenates of buffer-inoculated larvae and the PBS treatment.7.The influence of the inoculation of E.ludwigii on the regurgitation of caterpillarsTo test the potential effects of bacterial inoculation on altering the propensity for H.zea larvae to regurgitate on tomato leaves deserved examining,caterpillars inoculated with buffer or E.ludwigii were pre-fed on fluorescent dye-soaked artificial diet and then transferred onto tomato plants.Regurgitant collected from both buffer and E.ludwigii-inoculated larvae fed on dye-soaked diet were highly fluorescent,but no evident fluorescence spots were observed on tomato leaves damaged by larvae regardless of previous inoculation with E.ludwigii.8.Specific primers design and observation the existence of E.ludwigii on plantsThe specific primer sequence ENTITS3 designed from the 16S-23S rRNA intergenic transcribed spacer region(ITS)of E.ludwigii was highly specific and sensitive when used in combination with the universal primer EC5.Using this specific primer pair,we detected the presence of E.ludwigii only in regurgitant collected from E.ludwigii-inoculated caterpillars.The specificity of the primers was verified by sequencing the PCR products.However,E.ludwigii was not detected on feeding sites of tomato leaves damaged by E.ludwigii-inoculated caterpillars.