Functional Analysis Of Stress-associated Proteins And JmiC Domain-containine Histone Demethvlases In Disease Resistance Against Botrvtis Cinerea And Pst DC3000 In Tomato

Botrytis cinerea is a widespread plant fungal pathogen with a necrotrophic nutritional lifestyle.The fungus infects more than 200 monocots and dicot crop hosts.This polyphagous pathogen,which causes gray mold disease,is considered the second most important phytopathogen responsible for pre and postharvest decay and fruit quality deterioration in greenhouses,open fields and during storage.As a hemibiotrophic bacterial pathogen,Pseudomonas syringae pv.tomato DC3000(Pst DC3000)also causes economically important diseases in a wide range of plant species.In order to study tomato-B.cinerea or tomato-Pst DC3000 interaction,we adapted virus-induced gene silencing(VIGS)technology to explore the possible roles of tomato stress-associated protein(SAP)family and JmjC domain-containing protein(JMJ)family in defense resistance.In the study,we mainly focused on mechanisms of SlSAP3,SlSAP4 and SlJMJ7 in the resistance response to B.cinerea and Pst DC3000.SAPs have been reported to be involved in abiotic stress responses,including chilling,high temperature,water deficit,salinity,osmotic stress and heavy metals.Tomato SAP family,consisting of 13 members,most of which responded to Botrytis cinerea,Pst DC3000 and defense signaling hormones.VIGS-based knockdown of each of the SISAP genes and disease phenotyping assays indicated that silencing of SlSAN or SlSAP10 resulted in attenuated immunity to B.cinerea,whereas silencing of SlSAP3 or SISAP10 resulted in attenuated immunity to Pst DC3000.Silencing of SISAP10 exhibited an obvious accelerated yellowing phenotype and contained lower content of chlorophyll,thus we did not give further research,Functional analysis of SlSAP4 in decreased resistance against Botrytis cinerea reveals that silencing of SISAP4 increased accumulation of reactive oxygen species and downregulated expression of JA/ET signaling responsive defense genes after infection of B.cinerea.Transient expression of SlSAP4 in leaves of Nicotiana benthamiana led to enhanced resistance to B.cinerea.SlSAP4 did not show ubiquitin E3 ligase activity in vitro but interacted with three of four SlRAD23 proteins.Further biochemical analyses indicated that the A20 domain in SlSAP4 and the first UBA domain in SlRAD23d.1 were critical for the SlSAP4-SlRAD23d.1 interaction.Silencing of SlRAD23d.1 led to decreased resistance to B.cinerea but silencing of each of other three member of the SlRAD23 family did not affect the immunity against B.cinerea.These data suggest that SlSAP4 contributes positively to tomato immunity against B.cinerea through affecting the ROS balance and JA/ET signaling and may be involved in linking the substrate ubiquitination process with the ubiquitin/26S proteasome via interacting with SlRAD23s.Additional,VIGS and disease assays indicated that silencing of SlSAP3 resulted in attenuated immunity to Pst DC3000,whereas overexpressing SISAP3 increased immunity to Pst DC3000 accompanying with alerted expression of SA pathway defense genes after infection of Pst DC3000.Flg22-induced ROS burst and expression of PTI marker genes were strengthened in SISAP3-OE plants,whereas flg22-induced immune responses were suppressed in TRV-SlSAP3 plants.Although did not possess ubiquitin E3 ligase activity in vitro,SlSAP3 was found to interact with small heat shock proteins SIBOBs and ubiquitin receptors SlRAD23s.Further biochemical analyses indicate that the A20 domain in SlSAP3 and the UBA domain in SlRAD23d.1 are critical for the SlSAP3-SlRAD23d.1 interaction.Silencing of SIBOB1 or SIBOBa both led to increased resistance to Pst DC3000.Whereas silencing each of the SIRAD23 family members did slightly affect immunity to Pst DC3000.Taken together,these data suggest that SlSAP3 positively contributes to tomato immunity against Pst DC3000 and affects protein degradation process,possible through the ubiquitination/proteasome pathway by interacting with SIBOBs and SlRAD23s.Jumonji C(JmjC)domain-containing proteins have been characterized to be involved in histone demethylation.Growing evidences have shown that JmjCs epigenetically regulate various biological processes in plants.Here,we report the involvement of tomato SlJMJ family,consisting of 23 members in immunity.The SlJMJ genes responded with different expression patterns to Botrytis cinerea,Pst DC3000 and defense signaling hormones.Disease phenotyping assays indicated that silencing of SlJMJ7 or SlJMJ13 resulted in enhanced immunity to B.cinerea,whereas silencing of SlJMJ20 resulted in attenuated immunity to B.cinerea.Virus-induced SIJMJ7 gene silencing resulted in increased immunity to B.cinerea,accompanying with decreased reactive oxygen species(ROS)accumulation and suppressed and increased expression of SA and JA/ET signaling responsive defense genes,respectively after infection of B.cinerea.However,SlJMJ7-OE plants did not affect resistance to B.cinerea.On the other hand,SlJMJ7-silenced plants also enhanced resistance to Pseudomonas syringae pv.tomato DC3000,while its overexpression lines attenuated resistance through negatively modulating of several SA-related defense genes and two PTI-related genes(a tomato kinase PTI5 and a transcription factor,SlWRKY28).We demonstrated that SlJMJ7,containing a catalytic JmjC domain with conserved Fe(?)and alpha-ketoglutarate binding sites,displays H3K9me2/3 demethylase activity both in vitro and in vivo.Additionally,SlJMJ7 interacted with a LBD transcription factor SlLBD42,silencing of SlLBD42 led to increased resistance to B.cinerea but attenuated resistance to Pst DC3000.These data suggest that SlJMJ7 played a critical role in plant defense against Botrytis cinerea and Pst DC3000,and associatied with transcriptional regulators to control plant defense processes.