The Function Dissection Of The StRFP1 In Regulating Late Blight Resistance In Potato

Potato(Solanum tuberosum L.)is the fourth food crop in the world.It plays an important role in world food security.Potato late blight,caused by oomycete Phytophthora infestans(Mont.)de Bary,is the destructive disease for potato production worldwide.Systematic analysis of resistance mechanism is important to explore the management strategies for control late blight.Numerous studies have shown that ubiquitination is an important post-translational modification that plays a crucial role in regulating of plant immune responses,including PTI and ETI.Ubiquitination is a major posttranslational modification throught regulating protein stability.A number of RING type E3 s have been documented to be involved in plant immune signaling pathway.All the ATL family proteins contain the RING motif and four additional characteristic motifs: transmembrane domain(TM),basic amino acid-rich region,conserved GLD tri-peptide sequence and C-terminal diverse region.ATLs have been shown to participate in the regulation of the growth,development and biotic and abiotic stress.Previously,we have shown that a potato P.infestans and abiotic stress responsive ATL gene St RFP1 positively regulates late blight resistance.In this study,deeper investigation is conducted on the role of St RFP1 and it’s orthologue Nb ATL60 from Nicotiana benthamiana in immunity against P.infestans.1.St RFP1 and its orthologue Nb ATL60 both strongly responds to culture filtrate(CF)and the well-characterised bacterial PAMP flg22 induction,indicating that St RFP1 and Nb ATL60 involved in the activation of PTI in Solanaceous plants.2.Both St RFP1 and Nb ATL60 containe a transmembrane domain at the N-terminus of the protein.St RFP1: GFP and Nb ATL60: GFP were found to predominantly localize to plasma membrane and punctate vesicular structures by confocal microscopy.This indicated they might undergo constitutive endocytic trafficking.Confocal microscopy visulized that CF/culture medium and flg22/DMSO buffer infiltration do not affect St RFP1 and Nb ATL60 localization in plant cells.3.In the E3 ligase activity assay,St RFP137-262-HIS and Nb ATL6034-256(without the transmembrane domain)showed E3 ligase activity by forming a high-molecular-mass polyubiquitin ladder.St RFP137-262(H124A)and Nb ATL6034-256(H127A)had lost ligase activity.The results verified that both St RFP1 and Nb ATL60 have E3 ubiquitin ligase activity which dependents on an intact Ring-finger domain.4.St RFP1 overexpression(OE)potato lines enhanced P.infestans resistance and RNAi of St RFP1 in potato significantly reduced late blight resistance.Overexpression St RFP1-MYC and Nb ATL60-MYC in N.benthamiana significantly suppressed P.infestans growth.While overexpression of St RFP1-mut-MYC and Nb ATL60-mut-MYC,which losts E3 ubiquitin ligase activity,did not suppress P.infestans growth.This suggested that St RFP1 and Nb ATL60 act as positive regulators of immunity in N.benthamiana and their E3 ubiquitin ligase activity is essential for their function.5.In order to analyze the function of Nb ATL60,Virus-induced gene silencing(VIGS)was used to silence it in N.benthamiana.The TRV: ATL60 plants displayed a severe dwarf phenotype.Silencing of Nb ATL60 resulted in increased colonization of P.infestans indicating that it is essential for N.benthamiana to defend late blight.This results verified St RFP1 and Nb ATL60 have the same function for regulating late blight resistance.It had been reported that some ATLs regulated cell death in N.benthamiana leaves.Our results showed that silencing of Nb ATL60 does not affect cell death triggered by either P.infestans PAMP INF1 or any of the R protein and corresponding Avirulences protein pairs(Rx/Cp,R2/Avr2,R3a/Avr3 a,Pto/Avr Pto).6.q RT-PCR was used to detect four PTI marker genes including WRKY7/8,ACRE31 and Pti5 in St RFP1 overexpression(OE)and RNAi(Ri)potato lines and in Nb ATL60 VIGS N.benthamiana plants 0.5 h after flg22 treatment.All four PTI marker genes were upregulated in the OE potato line and down-regulated in the Ri potato line.The expression of the four marker genes exhibited the down-regulation pattern in N.benthamiana VIGS plants.Together,the above results indicated that St RFP1 and Nb ATL60 positively regulate PTI marker genes both in potato and N.benthamiana plants.7.In order to further explore how St RFP1 combined with target protein to regulate plant innate immunity,71 positive clones were identified using a plasma membrane Yeast Two-Hybrid(Y2H)system in this study.Through the yeast point-to-point verification,we found that 3 strong interaction proteins St?-GLU08,St HMA and St LD and two weak interaction proteins St ACY and St14-3-3.The yellow fluorescence complementation(Bi FC)test further verified that St ACY and St14-3-3 interacted with St RFP1.Overexpression of St ACY-MYC and St14-3-3-MYC in N.benthamiana significantly suppresses P.infestans growth.This suggests that St ACY and St14-3-3 act as positive regulators of immunity in N.benthamiana and implicated that the E3 ligase St RFP1 enhancing late blight resistance is not by degradation of St ACY and St14-3-3.Above results suggested that St RFP1 might need interaction proteins and other potential targets to regulate plant resistance.