A Phytophthora effector recruits a host cytoplasmic transacetylase into nuclear speckles to enhance plant susceptibility
Epigenetic modifications regulate numerous biological processes including growth and development and disease resistance ( Gómez-Díaz et al . , 2012 ) . Epigenetic modifications , including chromatin remodeling by acetylation , methylation , and ubiquitination of histones , affect the expression levels of defense genes upon pathogen attack ( Zhu et al . , 2016 ) . Several studies have indicated that epigenetic regulation can be triggered by plant-pathogen interactions . In Arabidopsis , the transcription factor WRKY70 , which activates salicylic acid ( SA ) -regulated defense-response genes , is epigenetically controlled through the trimethylation of lysine 4 of histone H3 ( H3K4me3 ) ( Alvarez-Venegas et al . , 2007 ) . Arabidopsis histone methyltransferases SET DOMAIN GROUP8 ( SDG8 ) and SDG25 regulate immune responses triggered by PAMPs ( pep1 and flg22 ) or effectors by modulating global histone lysine methylation ( Lee et al . , 2016 ) . Histone H2B monoubiquitination mediated by Arabidopsis HISTONE MONOUBIQUITINATION1 ( HUB1 ) is involved in protecting plants against necrotrophic fungi ( Dhawan et al . , 2009 ) . Histone acetylation is an important epigenetic modification involved in remodeling chromatin structure and regulating gene expression in eukaryotes ( Berger , 2007 ) . The levels of histone acetylation are regulated by histone acetyltransferases ( HATs ) and histone deacetylases ( HDACs ) ( Roth et al . , 2001 ) . Previous studies have shown that histone acetylation regulates plant immunity . For example , HDAC19 regulates resistance-gene expression in response to jasmonic acid and ethylene signaling during pathogen infection in Arabidopsis ( Zhou , 2005 ) . In rice , HDT701 , which deacetylates histone H4 of defense-related genes , negatively modulates plant immunity ( Ding et al . , 2012 ) . However , the potential mechanisms by which pathogen effectors might regulate epigenetic modifications by interacting with HATs and HDACs remain unclear . Some reports have indicated that effectors secreted by plant pathogens are involved in epigenetic modifications that manipulate plant immunity . The Phytophthora sojae effector PsAvh23 suppresses defense gene expression to promote infection of soybean by inhibiting acetylation of lysine 9 of histone H3 ( H3K9 ) ( Kong et al . , 2017 ) . Geminivirus TrAP inhibits histone methyltransferase SUVH4/KYP to decrease repressive H3K9me2 and DNA methylation , resulting in activation of viral virulence and plant susceptibility genes to attenuate host defense ( Castillo-González et al . , 2015 ) . The maize pathogen , Cochliobolus carbonum , produces a non-ribosomal peptide toxin , HC Toxin , that inhibits the histone deacetylase of its host , resulting in hyperacetylation of both histones and non-histone proteins ( Brosch et al . , 1995; Ransom and Walton , 1997; Walley et al . , 2018 ) . In the animal parasite , Toxoplasma , effectors block STAT1-dependent transcription by manipulating the histone deacetylase complex Mi-2/NuRD ( Olias et al . , 2016 ) . Although these cases suggest that pathogen manipulation of host epigenetic processes may be a broad virulence strategy , the full spectrum of underlying mechanisms remains unexplored . Filamentous fungi and oomycete pathogens secrete intracellular and extracellular effectors to manipulate host immunity ( Giraldo and Valent , 2013; Tyler et al . , 2006 ) . Oomycete-secreted RxLR effectors are cytoplasmic effectors that possess a conserved RxLR ( Arg-x-Leu-Arg ) motif to facilitate translocation from pathogens into host cells ( Kale et al . , 2010; Dou et al . , 2008 ) . However , the various mechanisms by which RxLR effectors manipulate plant immunity remain to be fully defined . Some plant targets of RxLR effectors have been identified that regulate plant immunity . For example , the P . infestans effector AVR3a manipulates plant immunity by stabilizing the host E3 ligase , CMPG1 ( Bos et al . , 2010 ) . The P . sojae effectors PSR1 and PSR2 suppress RNA silencing in immune signaling pathways in plants to promote Phytophthora infection ( Qiao et al . , 2013 ) . The Nudix hydrolase activity of PsAvr3b reduces host NADH and ADP-ribose levels ( Dong et al . , 2011 ) following activation by GmCYP1 ( Kong et al . , 2015 ) . PsAvh262 interacts with soybean proteins that control endoplasmic reticulum stress in order to modulate plant resistance ( Jing et al . , 2016 ) . PsAvr3c reprograms host pre-mRNA splicing to subvert plant immunity , to promote disease ( Huang et al . , 2017 ) . In our previous research , we found that the transcript levels of the RxLR effector gene PsAvh52 are strongly upregulated during the early stages ( 0 . 5 – 6 h . p . i . ) of P . sojae infection ( Wang et al . , 2011; Ma et al . , 2015 ) . Furthermore , PsAvh52 could suppress cell death induced by effectors and PAMPs in Nicotiana benthamiana ( Wang et al . , 2011 ) and soybean ( Ma et al . , 2015 ) . These results indicated that PsAvh52 could potentially suppress plant defense responses during the early stages of pathogen infection . In this study , we examined whether PsAvh52 is required for pathogenesis and the mechanism by which it suppresses plant immunity . We found that PsAvh52 was required for the full virulence of P . sojae , and promoted P . sojae infection when overexpressed in soybean hairy roots . PsAvh52 interacted with GmTAP1 , a novel acetyltransferase , causing GmTAP1 to relocate into the nucleus . In the nucleus , GmTAP1 acetylated core histones , up-regulating the expression of potential plant susceptibility genes . PsAvh52 targeted a different set of host genes than the effector that suppresses acetylation , PsAvh23 , and at an earlier time point than PsAvh23 .
Genome sequence analysis found that PsAvh52 is conserved in four primary P . sojae isolates ( Figure 1—figure supplement 1A ) . This indicated that PsAvh52 may be important for pathogenicity . To explore the role of PsAvh52 in pathogenesis , we deleted the PsAvh52 gene from P . sojae using CRISPR/Cas9 ( Fang and Tyler , 2016 ) . Based on genomic DNA PCR analysis , we identified two knockout transformants ( T33 and T37 ) ( Figure 1—figure supplement 1B , C ) . There was no growth difference in vitro between WT , CK and ∆PsAvh52 transformants ( Figure 1—figure supplement 1D , E ) . However , the two knockout transformants produced smaller lesions on soybean seedlings compared to WT and CK ( Figure 1A ) . Furthermore , the biomass of P . sojae was lower in soybean seedlings infected by transformants T33 and T37 than by WT and CK ( Figure 1B ) . These results indicate that PsAvh52 is required for the full virulence of P . sojae . To further test whether PsAvh52 could promote P . sojae infection , GFP-PsAvh52 ( without a signal peptide ) was transiently expressed in soybean hairy roots using Agrobacterium rhizogenes ( K599 ) -mediated transformation . The soybean hairy roots were inoculated with P . sojae P6497 labeled by the red fluorescent protein mRFP . Compared to the EV control , expression of GFP-PsAvh52 in soybean hairy roots promoted P . sojae infection 48 hr post-inoculation ( hpi ) ( Figure 1C–E ) . These data indicate that PsAvh52 is a required virulence factor that can enhance the susceptibility of soybean to P . sojae infection . To identify host target proteins of PsAvh52 , we performed co-immunoprecipitation ( Co-IP ) assays . We transiently expressed GFP-tagged PsAvh52 in soybean hairy roots and purified PsAvh52 and its potential plant targets using GFP affinity beads . These proteins were identified by liquid chromatography tandem-mass spectrometry ( LC-MS/MS ) . Several proteins were identified that potentially associate with PsAvh52 ( Figure 2—source data 1 ) . One of these was a putative soybean transacetylase protein , which we designated as GmTAP1 . Three isoforms were predicted to be encoded by the GmTAP1 gene ( Figure 2—figure supplement 1 ) . Of these three , however , we could only clone the longest isoform of GmTAP1 ( Glyma . 18G216900 . 1 ) from soybean cDNA . A predicted paralog of GmTAP1 , GmTAP2 ( Glyma . 09G272400 . 1 ) , shared around 85% amino acid identity according to the Phytozome genome database ( Figure 2—figure supplement 1 ) . To examine the specific interactions of PsAvh52 with GmTAP1 and GmTAP2 , we performed Co-IP experiments . Flag-PsAvh52-RFP was transiently co-expressed with GFP-GmTAP1 , GFP-GmTAP2 , or GFP in N . benthamiana , after which the proteins were incubated with GFP affinity beads . As shown by western blotting , Flag-PsAvh52-RFP was co-immunoprecipitated with GFP-GmTAP1 , but not with GFP-GmTAP2 or GFP ( Figure 2A ) . In addition , glutathione S-transferase ( GST ) pull-down assays were performed to confirm the interaction between PsAvh52 and GmTAP1 or GmTAP2 in vitro . The recombinant GST-GmTAP1 and GST-GmTAP2 proteins were purified from Escherichia coli with GST beads and incubated with His-PsAvh52 . His-PsAvh52 protein was pulled down by GST-GmTAP1 , but not by GST-GmTAP2 or GST alone ( Figure 2B ) . To map the key region that is required for PsAvh52 interaction with GmTAP1 , we constructed a series of deletion mutations and identified a mutant PsAvh52M4 ( deletion of amino acids 69 – 86 ) that could not associate with GmTAP1 ( Figure 2C , Figure 2—figure supplement 2A , D , E ) , demonstrating that the region of amino acids 69 – 86 is essential for the PsAvh52 interaction with GmTAP1 . When PsAvh52M4 was expressed in soybean hairy roots , it could not promote P . sojae infection compared to PsAvh52 , indicating that PsAvh52M4 lost its virulence-promoting function ( Figure 2D , E ) . These results suggest that the amino acids 69 – 86 of PsAvh52 are essential for the interaction with GmTAP1 and promoting infection . To determine the subcellular localization of GmTAP1 , we expressed GmTAP1 fused to N-terminal GFP ( GFP-GmTAP1 ) or C-terminal RFP ( GmTAP1-RFP ) using Agrobacterium-mediated transient expression in N . benthamiana and observed the subcellular localization of GmTAP1 by confocal microscopy . Confocal microscopy showed that both GFP-GmTAP1 and GmTAP1-RFP localized in the cytoplasm , but not in the nucleus ( Figure 3—figure supplement 1A ) . To evaluate the interaction of PsAvh52 with GmTAP1 , we observed the localization of PsAvh52 and GmTAP1 during transient co-expression in N . benthamiana . Interestingly , when GFP-GmTAP1 and PsAvh52-RFP were co-expressed in N . benthamiana , GmTAP1 strongly localized in the nucleus as well as in the cytoplasm . In the nucleus it co-localized with PsAvh52 in speckles after 24 hr expression ( Figure 3A ) and throughout the nucleus after 36 – 48 hr expression ( Figure 3—figure supplement 1B , C ) . To confirm this localization pattern , we changed the fluorescent proteins and their fusion positions on PsAvh52 and GmTAP1 . When GFP-PsAvh52 and GmTAP1-RFP were co-expressed in N . benthamiana , green and red fluorescent signals were detected in the nucleus and cytoplasm; again the proteins were localized to nuclear speckles after 24 hr ( Figure 2—figure supplement 2A ) but throughout the nucleus after 36 – 48 hr ( Figure 3—figure supplement 1B , C ) . Western blot analysis showed that GmTAP1 was present in the nuclear protein fraction only when co-expressed with PsAvh52 ( Figure 3B ) . These data suggested that PsAvh52 could cause the relocation of GmTAP1 into the nucleus when the two proteins were co-expressed in N . benthamiana . The localization pattern of GmTAP1 during P . sojae infection of soybean could not be tested , however , because no soybean hairy roots accumulating GFP-GmTAP1 protein could be identified , when the GFP-GmTAP1 overexpression construct was used . The nuclear localization signal ( NLS ) is an important amino acid motif for import of proteins into the cell nucleus ( Kalderon et al . , 1984 ) . It possesses one or more positively charged lysine or arginine residues , which are important for its function ( Makkerh et al . , 1996 ) . A potential NLS was identified in PsAvh52 using the Simple Modular Architecture Research Tool ( www . moseslab . csb . utoronto . ca/NLStradamus ) . To confirm the function of the predicted NLS and to explore whether the NLS was important for the biological function of PsAvh52 , we mutated all of the lysine residues of the NLS to alanine , generating the mutant PsAvh52M1 ( Figure 2—figure supplement 2A ) . The wild-type PsAvh52 was localized to the nuclear speckles , but mutant PsAvh52M1 was not localized to the nucleus at all , confirming the function of the predicted NLS ( Figure 2—figure supplement 2F ) . Then we observed the localization of GmTAP1-RFP when co-expressed with GFP-PsAvh52M1 in N . benthamiana . In this case , neither GmTAP1 nor PsAvh52M1 were localized in the nucleus ( Figure 2—figure supplement 2A ) . These results indicate that the nuclear localization of PsAvh52 was required for relocation of GmTAP1 into the nucleus . Compared to PsAvh52 , the susceptibility-promoting activity of PsAvh52M1 in soybean hairy roots was no higher than the GFP control ( Figure 2—figure supplement 2B ) . These results show that the nuclear localization of PsAvh52 is required for its virulence contribution and for its ability to cause GmTAP1 to relocate into the nucleus . As PsAvh52 is a nucleus-localized effector with an NLS , we explored whether other effectors with an NLS ( PsAvh53 , PsAvh137 ) could cause GmTAP1 to relocate into the nucleus . GmTAP1-RFP and GFP-effector fusions were co-expressed in N . benthamiana , but red fluorescent signal was never detected in the nucleus , indicating that these other effectors with an NLS could not affect the localization of GmTAP1 ( Figure 3—figure supplement 1D–F ) . These data suggested that PsAvh52 causes GmTAP1 to relocate into the nucleus through a specific interaction . To test if direct interaction between GmTAP1 and PsAvh52 was required for the relocation of GmTAP1 into the nucleus , the GmTAP1-non-interacting mutant PsAvh52M4 was tested for its ability to cause GmTAP1 to relocate . As shown in Figure 3 , PsAvh52M4 could not cause GmTAP1 to relocate into the nucleus when the two were transiently co-expressed in N . benthamiana . These results suggest that the amino acids 69 – 86 of PsAvh52 , that are essential for the interaction , are also required for its ability to trigger the relocation of GmTAP1 into the nucleus . Interestingly , deletion of the interacting region caused PsAvh52 to no longer localize to the nuclear speckles; instead it remained in the nucleoplasm and the nucleolus ( Figure 2—figure supplement 2F ) . To determine which region of GmTAP1 was targeted by PsAvh52 , we split GmTAP1 into two parts: the N-terminal region , GmTAP11-140 , and the C-terminal region , GmTAP1atd , containing the predicted acetyltransferase domain ( ATD ) . We expressed GFP-GmTAP1 , GFP-GmTAP11-140 , GFP-GmTAP1atd , and GFP in N . benthamiana leaves and purified them using GFP affinity beads . We used the purified GFP-proteins in pull-down assays with His-PsAvh52 purified from E . coli . His-PsAvh52 co-precipitated with GFP-GmTAP1 and GFP-GmTAP11-140 , but not with GFP-GmTAP1atd nor with GFP ( Figure 3—figure supplement 2A ) . This result showed that the N-terminal region of GmTAP1 interacted with PsAvh52 . To confirm this interaction , we observed the localization of GFP-GmTAP11-140 by confocal microscopy , and found that in the absence of PsAvh52 it localized to the cytoplasm , and was excluded from the nucleus , similar to GFP-GmTAP1 ( Figure 3—figure supplement 2B ) . In contrast , GFP-GmTAP1atd localized not only to the cytoplasm but also strongly and uniformly to the nucleoplasm ( Figure 3—figure supplement 2B ) , suggesting that the N-terminal domain is responsible for regulating the nuclear and sub-nuclear localizations of the C-terminal acetyltransferase domain , even in the absence of PsAvh52 . When PsAvh52-RFP was co-expressed with GFP-GmTAP11-140 or GFP-GmTAP1atd in N . benthamiana , GmTAP11–140 re-localized from the cytoplasm to nuclear speckles together with PsAvh52-RFP after 24 hr expression and throughout the nucleus after 36 – 48 hr expression , but the localization of GFP-GmTAP1atd was not affected by the presence of PsAvh52-RFP ( Figure 3—figure supplement 2B , C ) . Furthermore , expression of GmTAP1atd in N . benthamiana could not promote P . capsici infection ( Figure 3—figure supplement 2E ) . Together these results indicate that interaction of the N-terminal region of GFP-GmTAP11–140 with PsAvh52 was required for the relocalization of GFP-GmTAP1 into the nucleus in the presence of the effector . Given that PsAvh52 interacted with and caused the relocalization of GmTAP1 into the nuclear speckles , we directly examined the role of GmTAP1 in plant immunity . To this end , we silenced the GmTAP1 gene in soybean hairy roots using a hairpin RNAi construct ( Xiong et al . , 2014 ) . Because the gene sequences of GmTAP1 and GmTAP2 are conserved , it was difficult to silence GmTAP1 alone . We designed primers specific to the 3’ UTR region in an attempt to specifically silence the GmTAP1 gene , but the two GmTAP genes were still both silenced . The silencing constructs were transformed into soybean hairy roots via agroinfiltration . Compared to EV-transformed hairy roots , the transcript levels of the two GmTAP genes were significantly decreased in the GmTAP-silenced hairy roots ( Figure 4B ) . To explore the effects of GmTAP-silencing on soybean resistance to P . sojae , GmTAP-silenced hairy roots were challenged with P . sojae labeled with mRFP . The number of oospores produced was lower in GmTAP-silenced hairy roots than in hairy roots carrying EV ( Figure 4A , C ) . To confirm these results , we used quantitative real time PCR ( qPCR ) to determine the biomass of P . sojae in the soybean hairy roots . When GmTAP-silenced hairy roots were inoculated with WT P . sojae , the P . sojae biomass was only 40% of that when WT was inoculated onto control EV-carrying hairy roots ( Figure 4D ) . To confirm that GmTAP1 acts as a true virulence target of PsAvh52 , the GmTAP-silenced hairy roots were challenged with the P . sojae PsAvh52 deletion mutant , T37 . When GmTAP-silenced hairy roots were inoculated with T37 , the P . sojae biomass was no less than when T37 was inoculated onto control EV-carrying hairy roots ( Figure 4D ) . These results show that the ability of GmTAP1 to promote susceptibility to P . sojae depends on the expression of PsAvh52 in the pathogen . The P . sojae biomass of T37 infecting GmTAP-silenced hairy roots was nearly the same as WT infection of the same roots ( Figure 4D ) , indicating that the virulence-promoting activity of PsAvh52 is primarily dependent on the expression of GmTAP1 . It is not clear if the small but statistically significant difference between T37 and WT is due to incomplete GmTAP silencing , additional functions of PsAvh52 , other strain differences , or is not biologically relevant . As GmTAP2 was silenced in the RNAi experiments , we did several experiments to check the contribution of GmTAP2 to immunity . First , the localization of GmTAP2 was observed in N . benthamiana . We found that GmTAP2 was localized in both the plant cell nucleus and cytoplasm , which was different from the localization of GmTAP1 and resembled the localization of GmTAP1atd ( Figure 3—figure supplement 2B , Figure 4—figure supplement 1A ) . As expected ( since PsAvh52 and GmTAP2 do not interact physically ) , when GFP-GmTAP2 and PsAvh52-RFP were co-expressed in N . benthamiana , GmTAP2 localization was not affected by the presence of PsAvh52 ( Figure 4—figure supplement 1B ) . Furthermore , expression of GmTAP2 in N . benthamiana could not promote P . capsici infection ( Figure 4—figure supplement 1C–E ) , unlike GmTAP1 ( see next section ) . These results suggested that GmTAP2 probably did not contribute to plant immunity . In the experiments described above , we showed that PsAvh52 caused the relocation of GmTAP1 into nuclear speckles , and that the relocation of GmTAP1 into the nuclear speckles was associated with promotion of susceptibility to P . sojae . There could be two hypotheses regarding GmTAP1 relocalization into the nuclear speckles: ( 1 ) GmTAP1 acts as a positive regulator of immunity when localized in the cytoplasm , and relocalization into the nuclear speckles abolishes this function; ( 2 ) GmTAP1 does not affect plant immunity when localized in the cytoplasm , but acts as a negative regulator of immunity when localized in the nuclear speckles . To test these hypotheses , we inoculated P . capsici onto N . benthamiana leaves expressing various versions of GmTAP1 . P . capsici does not contain any homologs of PsAvh52 . Compared with leaves expressing the control GFP , native GmTAP1 , which localized to the cytoplasm but not the nucleus ( Figure 5—figure supplement 1A ) , did not affect susceptibility to P . capsici infection ( Figure 5A , B ) . On the other hand , transient expression of GmTAP1 fused to an NLS ( NLS-GmTAP1 ) , which predominantly localized into nuclear speckles , did increase susceptibility to P . capsici infection . In contrast , transient expression of GmTAP1 fused to a nuclear export signal ( NES-GmTAP1 ) , which ensured that GmTAP1 remained in the plant cytoplasm , did not promote infection ( Figure 5A , B; Figure 5—figure supplement 1A , B ) . These results indicated that GmTAP1 localization into the plant nucleus is sufficient to promote plant susceptibility to Phytophthora infection . Since it was predicted that GmTAP1 possesses GNAT family N-acetyltransferase enzyme activity ( Smith et al . , 1998; Dutnall et al . , 1998 ) , we tested whether GmTAP1 could acetylate core histones in vitro . First , GST-GmTAP1 was expressed and purified in E . coli ( Figure 5—figure supplement 1C ) . Using chicken core histones as substrates , we found that GmTAP1 had histone acetyltransferase ( HAT ) activity . GmTAP1 could acetylate lysine 9 of histone H3 and lysines 5 and 9 of histone H2A ( Figure 5C ) . In addition , C646 ( a HAT inhibitor ( Bowers et al . , 2010 ) ) could inhibit the enzyme activity of GmTAP1 . We further tested whether PsAvh52 affected the HAT activity of GmTAP1 . His-PsAvh52 protein was purified from E . coli and incubated with GST-GmTAP1 , after which we measured the enzyme activity . We found that the HAT activity of GmTAP1 was not affected after incubation with His-PsAvh52 ( Figure 5C ) . Thus , PsAvh52 did not affect the enzyme activity of GmTAP1 , consistent with its interaction with the N-terminal non-enzymatic region of GmTAP1 ( Figure 3—figure supplement 2A ) . There are five key amino acid residues ( 167-169 , 179-180 ) predicted to be required for HAT activity in GmTAP1 . We mutated those amino acid residues to alanine and confirmed experimentally that this mutant , GmTAP1-M1 , had lost HAT activity ( Figure 5C ) . Next , we explored the relationship between GmTAP1’s ability to increase plant susceptibility and its enzyme activity . We transiently expressed NLS-GmTAP1-M1 or NLS-GmTAP1 in N . benthamiana leaves then compared P . capsici infection of leaves expressing the two constructs . As shown in Figure 5A , the mutant failed to increase plant susceptibility to P . capsici as compared to NLS-GmTAP1 ( Figure 5A , B ) . We confirmed the localization of NLS-GmTAP1-M1 into nuclear speckles , similar to NLS-GmTAP1 ( Figure 5—figure supplement 1A ) . These results confirmed that the enzyme activity of GmTAP1 was required for its contribution to susceptibility . In order to test the histone transacetylase activity of GmTAP1 in planta , we transiently expressed GFP , GmTAP1 , NLS-GmTAP1 , NES-GmTAP1 , and NLS-GmTAP1-M1 in N . benthamiana leaves . The nuclear proteins were then extracted and the global histone acetylation levels were evaluated . As shown in Figure 5D , NLS-GmTAP1 increased the acetylation levels of histones H2A ( lysines 5 and 9 ) and H3 . In contrast , GmTAP1 lacking an NLS , GmTAP1 carrying an NES , and NLS-GmTAP1M1 did not increase acetylation levels of those histones ( Figure 5D ) . Furthermore , expression of GmTAPatd in N . benthamiana leaves also did not increase acetylation levels of those histones ( Figure 3—figure supplement 2D ) . This result indicated that N-terminus of GmTAP1 is important for targeting of the C-terminal enzymatic domain into the nuclear speckles , which is required for GmTAP1 to acetylate histones . The experiments described in the previous section demonstrated that histone acetylation was enhanced when GmTAP1 localized to the nucleus in N . benthamiana . To confirm that PsAvh52-mediated relocation of GmTAP1 into the nucleus increased histone acetylation in soybean , we measured acetylation of histones H2A and H3 in soybean hairy roots expressing either PsAvh52 or the GmTAP1-non-interacting mutant , PsAvh52M4 . After P . sojae infection for 6 hr , nuclear H2AK5 and H3K9 acetylation were increased in PsAvh52-expressing soybean hairy roots , but the expression of PsAvh52M4 did not affect the level of H2AK5 and H3K9 acetylation ( Figure 6A ) . Silencing of GmTAP1 in soybean hairy roots followed by P . sojae infection for 6 hr decreased H2AK5 and H3K9 acetylation relative to the EV-carrying control roots ( Figure 5—figure supplement 1D ) , suggesting that GmTAP1 is required for PsAvh52 to increase histone acetylation . To probe the ability of PsAvh52 to modify global acetylation levels of H2AK5 and H3K9 during P . sojae infection , we measured histone acetylation during infection of soybean hypocotyls ( 0 – 24 hr ) by WT P . sojae and by the PsAvh52 deletion mutant T37 ( Figure 6B ) . During early-stage ( 0 – 9 hr ) infection by WT , both H3K9 and H2AK5 acetylation increased strongly , but no increase was observed during early-stage infection by T37 ( Figure 6B ) . In a previous report , we found that during P . sojae infection , H3K9 acetylation increased first ( 0 – 6 hr ) and subsequently decreased ( 12 – 24 hr ) ( Kong et al . , 2017 ) . This result aligns with the expression profile of PsAvh52 , which showed higher expression during infection at 0 . 5 – 6 hr , suggesting that PsAvh52 acts primarily during early infection ( Ma et al . , 2015 ) . H2AK5ac and H3K9ac are epigenetic modifications associated with gene transcriptional activation ( Berger , 2007 ) . Since PsAvh52 promotes susceptibility through GmTAP1-mediated histone acetylation , it is plausible that PsAvh52 might cause an increase in expression of susceptibility genes . To test this hypothesis , we examined the expression of soybean orthologs of several reported susceptibility genes that play a role during pathogen-host interactions ( van Schie and Takken , 2014 ) . Using qRT-PCR , we measured the expression levels of soybean orthologs of a SWEET gene ( MtN3 , Glyma . 08G010000 ) ( Oliva and Quibod , 2017; Gebauer et al . , 2017 ) , a cell wall-degrading polygalacturonase ( PG , Glyma . 08G287500 ) ( Nühse , 2012; Cantu et al . , 2008 ) , and two lipoxygenases ( LOX-1 , Glyma . 13G347800 , LOX-2 , Glyma . 08G189200 ) ( van Schie and Takken , 2014; Gao et al . , 2007 ) . These putative susceptibility genes were upregulated in PsAvh52-expressing soybean hairy roots after 6 hr of P . sojae infection , but downregulated in GmTAP1-RNAi soybean hairy roots at the same time point ( Figure 6C ) . A control gene ( Ubiquitin 5; UBQ5 ) showed no significant change in expression level in response to either treatment . To examine changes in histone acetylation that may be associated with gene transcriptional activation , we performed chromatin immunoprecipitation ( ChIP ) with H2AK5ac and H3K9ac antibody , followed by qPCR ( ChIP-qPCR ) . Increased acetylation of histones of the four putative susceptibility genes was observed in PsAvh52-expressing soybean hairy roots , compared to empty vector transformed hairy roots , after 6 hr of P . sojae infection . In contrast , the control gene ( UBQ5 ) showed no significant change in histone acetylation ( Jaskiewicz et al . , 2011 ) ( Figure 6D ) . These results are consistent with a model in which PsAvh52 acts to increase the transcript levels of key susceptibility genes .
Increasing evidence suggests that epigenetic modifications including histone acetylation are an important mediator of plant defenses during plant-pathogen interactions ( Zhu et al . , 2016; Alvarez et al . , 2010 ) . However , the ability of plant pathogens to use effectors to disturb or co-opt host epigenetic modifications to promote infection has not been thoroughly explored . In this study , we found that the P . sojae effector PsAvh52 , expressed in soybean hairy roots , promoted P . sojae infection . PsAvh52 could bind to the soybean acetyltransferase , GmTAP1 , causing it to re-localize from the cytoplasm into nuclear speckles , a process that required the PsAvh52 NLS as well as protein-protein interactiondomains on both PsAvh52 and GmTAP1 . Furthermore GmTAP-silencing in soybean hairy roots by RNAi reduced susceptibility to P . sojae . Finally , when GmTAP1 was localized into the nuclear speckles , it could increase the level of histone H2AK5 and H3K9 acetylation and enhanced plant susceptibility , both of which required the histone acetyltransferase enzyme activity of GmTAP1 . Together , these results indicate that PsAvh52 recruits GmTAP1 to promote plant susceptibility through epigenetic modifications . HATs include two broad classes based on their subcellular localization; namely , nuclear localization and cytoplasmic localization . The cytoplasmic HATs acetylate newly synthesized histones prior to their assembly into nucleosomes ( Lee and Workman , 2007; Boycheva et al . , 2014 ) . Although we only observed cytoplasmic localization by GmTAP1 in the absence of PsAvh52 , several pieces of evidence suggest that it may be targeted to the nucleus under certain conditions . First , it belongs to the GNAT family of nuclear-targeted histone acetylases ( Lee and Workman , 2007; Boycheva et al . , 2014 ) . Second , it acetylated histones H2A and H3 , whereas canonical cytoplasmic HATs are specific for histone H4 ( Lee and Workman , 2007; Boycheva et al . , 2014 ) . Third , when it was targeted to the nucleus by addition of an NLS , it displayed a specific sub-nuclear localization ( nuclear speckles ) suggesting that it had natural targets in the nucleus . Fourth , when its N-terminal PsAvh52-interaction domain was removed , it localized substantially into the nucleus , but it did not localize in the nuclear speckles nor could it promote Phytophthora infection . Thus , the N-terminal domain may regulate the sub-cellular localization of GmTAP1 , excluding it from the nucleus as the default state , but targeting it to the nuclear speckles when it is transported into the nucleus . Together these results suggest that , under certain physiological conditions and/or in certain tissues , GmTAP1 may relocate into the nuclear speckles to modify histone acetylation , and that relocation into the nucleus may be regulated by proteins that interact with the N-terminal domain . If this is true , then PsAvh52 has evolved to subvert the normal process by which the activity of GmTAP1 activity is regulated . PsAvh52 localized to the nuclear speckles and this localization required the M4 GmTAP1-interaction domain . However , since N . benthamiana contains a GmTAP1 homolog , our data do not reveal whether PsAvh52 has an intrinsic speckle-targeting capability , or whether it relies on binding to GmTAP1 to target the speckles . Our current model is that PsAvh52 physically recruits GmTAP1 into the nucleus as a protein-protein complex , and that once in the nucleus , the complex is targeted to nuclear speckles through speckle-targeting signals on GmTAP1 at least , and perhaps on PsAvh52 also . However , since we did not identify a protein complex containing nuclear importins , PsAvh52 and GmTAP1 ( and did not try to ) , we cannot rule out other mechanisms that do not involve PsAvh52 and GmTAP1 entering the nuclear as a physical complex . Nuclear speckles have most often been described as complexes involved in pre-mRNA processing ( Galganski et al . , 2017 ) . Our data do not indicate whether the speckles targeted by PsAvh52 and GmTAP1 are also involved in pre-mRNA processing or whether we have observed a new class of speckles . We note that extended over-expression of PsAvh52 and GmTAP1 for 36 hr to 48 hr in N . benthamiana cells resulted in an apparent uniform distribution of the two proteins throughout the nucleus rather than concentration in the nuclear speckles . Our data do not distinguish whether the nuclear speckles had become saturated with GmTAP1-PsAvh52 resulting in the excess complex accumulating in the nucleoplasm , or whether the complexes dissociated from the speckles over time . Many nuclear speckles are dynamic structures and proteins associated with nuclear speckles may diffusely distribute throughout the nucleoplasm ( Sleeman et al . , 1998 ) . GmTAP2 showed 85% sequence identity with GmTAP1 in soybean , differing primarily in the N-terminus . When expressed in N . benthamiana , GmTAP2 localized to the cytoplasm and also to the nucleus , but did not localize to nuclear speckles in either the presence or absence of PsAvh52 . Furthermore , GmTAP2 did not bind to PsAvh52 and expression of GmTAP2 in N . benthamiana did not affect plant immunity . In addition , similar to GmTAP2 , expression of GmTAP1atd which also localized to the cytoplasm and nucleus did not affect histone acetylation or plant immunity in N . benthamiana . These observations are consistent with our model that in order to promote susceptibility , GmTAP1 must be targeted to certain nuclear speckles via the concerted action of the PsAvh52 and the N-terminal domain of GmTAP1 . The closest homolog of GmTAP1 in Arabidopsis is the nuclear shuttle protein-interacting protein ( AtNSI ) ( McGarry , 2003 ) . AtNSI was identified as binding to the nuclear shuttle protein ( NSP ) of cabbage leaf curl geminivirus and as having the ability to acetylate histones H2A and H3 ( McGarry , 2003 ) , consistent with our findings . McGarry , 2003 did not observe that AtNSI could activate transcription , but used only an in vitro assay for this test . They proposed that AtNSI may regulate the ability of NSP to regulate the shuttling of geminivirus DNA from the nucleus to the cytoplasm ( McGarry , 2003 ) . Thus GmTAP1/AtNSI may have been co-opted by two very different pathogens . GmTAP1 re-localization from cytoplasm to nucleus is a significant effect of the presence of PsAvh52 . The manipulation of host protein localization by effectors is an emerging theme in pathogen virulence mechanisms . Two other P . sojae effectors , PsCRN63 and PsCRN115 , could recruit the catalase NbCAT1 into the plant nucleus to perturb H2O2 homeostasis , and promote Phytophthora infection ( Zhang et al . , 2015 ) . The Phytophthora infestans effector Pi03192 prevented the transport of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus during infection , which promoted disease progression ( McLellan et al . , 2013 ) . Another P . infestans effector , Pi04314 , could bind to the host protein phosphatase one catalytic subunit ( PP1c ) , causing it to re-localize from the nucleolus to the nucleoplasm , promoting P . infestans infection ( Boevink et al . , 2016 ) . The P . infestans effector AVRblb2 , bound to plant protease C14 at the haustorial interface , preventing its secretion ( Bozkurt et al . , 2011 ) . Our finding that PsAvh52 caused GmTAP1 to relocate into host nuclear speckles to acetylate histones and promote susceptibility adds to this growing theme . Previous studies have shown that protein acetylation plays a key role in host-pathogen interactions ( Gómez-Díaz et al . , 2012; Alvarez et al . , 2010; Robert McMaster et al . , 2016 ) . Some pathogens enhance both host histone and non-histone protein acetylation to alter host immunity by secreting effectors that have acetyltransferase enzyme activity or that modulate host acetylases or deacetylases during a susceptible interaction . For example , HC toxin secreted by the fungal pathogen Cochliobolus carbonum inhibits maize histone deacetylase ( HD ) and increases the acetylation level of H3 and H4 , which may affect transcription at specific gene promoters ( Brosch et al . , 1995; Ransom and Walton , 1997; Walley et al . , 2018 ) . Here we showed that PsAvh52 could cause GmTAP1 to relocate into nuclear speckles to enhance global acetylation of H2AK5 and H3K9 during early P . sojae infection . Recently , Kong et al . , 2017 showed that another P . sojae effector , PsAvh23 , attenuated the acetylation of H3K9 by binding to the ADA2 subunit of the histone acetyltransferase complex , SAGA . Examination of the timing of expression of PsAvh52 and PsAvh23 , and the levels of H3K9 acetylation during infection by wild type P . sojae , and by PsAvh52 and PsAvh23 knockout strains , suggests that these two effectors may coordinately manipulate H3K9 acetylation levels to optimally promote P . sojae infection . PsAvh52 transcript levels peak at 1 hr post infection and then decline steadily over the next 11 to 23 hr ( Figure 6—figure supplement 1A; Ma et al . , 2015 ) . On the other hand , transcript levels of PsAvh23 are initially very low and do not rise substantially until after 6 hr ( Figure 6—figure supplement 1A; Wang et al . , 2011 ) . During the period from 6 to 12 hr , the transcript levels of PsAvh52 and PsAvh23 cross over ( Figure 6—figure supplement 1A ) . During wild type P . sojae infection , the global acetylation levels of H2AK5 and H3K9 first increase ( 0 – 9 hr ) and then sharply fall ( after 12 hr ) . However , during infection by P . sojae strains carrying a PsAvh23 deletion ( and a functional PsAvh52 gene ) , H3K9 acetylation level rise rapidly during infection , but never fall after 9 hr ( Figure 6—figure supplement 1B; Kong et al . , 2017 ) . In contrast , during infection by a PsAvh52 deletion strain ( carrying a functional PsAvh23 genes ) , H3K9 acetylation levels never rise , but remain at levels typical of late infection , and there is no further drop after 9 hr ( Figure 6B; Figure 6—figure supplement 1B ) . Overexpression of PsAvh23 or silencing of its target gene , GmADA2 , strongly suppressed the expression of multiple defense genes including ones encoding transcription factors and MAP kinase kinase kinases ( Kong et al . , 2017 ) . However , none of those genes were affected by over-expression of PsAvh52 or by silencing of GmTAP1 ( Figure 6—figure supplement 2 ) . On the other hand , several putative susceptibility genes were elevated by overexpression of PsAvh52 or silencing of GmTAP1 ( Figure 6 ) . Together , these observations suggest a model in which PsAvh52 acts early in infection to promote H3K9 acetylation , stimulating the expression of susceptibility genes , and then PsAvh23 acts to reverse the action of PsAvh52 , and also to block any host-mediated increases in H3K9 acetylation that may support the expression of plant defense genes . It will be of interest to determine if there are additional mechanisms , other than timing of expression , that determine which host genes are targeted by the actions of these two effectors .
The PsAvh52 gene and other P . sojae RxLR genes were cloned without their signal peptide coding regions from cDNA of P6497 . GmTAP genes were cloned from cDNA of soybean cultivar Williams 82 . All the constructs were cloned by homologous recombination with 15 – 20 bp of vector sequences at the 5’ terminus of each primer using the ClonExpress II One Step Cloning Kit ( Vazyme Biotech , C112 ) . PsAvh52 and GmTAP gene fragments were ligated into pBINGFP2 and pICH86988-RFP , which were used to express or localize proteins in plant cells . pEGX4T-2 and pET32a vectors were used to express proteins in E . coli for purification . PFGC5941 vector was used for gene silencing experiments in soybean hairy roots . N . benthamiana was cultivated at 24°C and soybean was cultivated at 25°C with 16 hr light and 8 hr darkness in humid conditions . P . sojae and P . capsici were cultivated at 25°C on 10% vegetable ( V8 ) juice agar medium . After soybean seeds were sterilized for 4 – 6 hr in chlorine gas , they were grown in moist vermiculite . After 5 – 6 days , soybean cotyledons were collected and sterilized with 10% hypochlorous acid ( HClO ) for 8 min , then in 70% ethanol for 40 s , then rinsed with sterile water several times . Then a wound was cut into each cotyledon and 20 μl of a suspension of K599 cells ( OD600 = 0 . 5 ) carrying the plasmid of interest was added to each wound . The inoculated soybean cotyledons were grown in 1/2 MS medium at 22°C with 16 hr light and 8 hr darkness . After 3 weeks cultivation , when soybean hairy roots had emerged , roots expressing green fluorescent protein were selected and tested for the expression of the genes of interest before inoculation with P . sojae . Recombinant vectors were introduced into Agrobacterium tumefaciens strain GV3101 by transformation . The transformed cells were incubated in LB medium with appropriate antibiotics at 28°C at 200 rpm for 24 – 36 hr . The bacteria were collected and washed with the infiltration buffer ( 10 mM MgCl2 , 10 mM MES , pH 5 . 7 , and 100 µM acetosyringone ) three times . After that , the bacteria were resuspended in infiltration buffer and adjusted to the required concentration before infiltration into attached leaves of 4 – 5 week-old N . benthamiana plants . After inoculation , the leaves were incubated at 25°C in the greenhouse for 24 hr to 72 hr depending on the purpose . For P . capsici infection assays on agroinfiltrated N . benthamiana leaves , the leaves were collected 48 hr after infiltration with A . tumefaciens , then inoculated with 10% V8 juice agar plugs ( diameter = 0 . 5 cm ) containing freshly grown P . capsici LT263 mycelia . After 36 hr - 48 hr inoculation , the P . capsici lesions were measured and photographed under a UV lamp . For P . sojae infection assays on soybean hypocotyls , mycelia were washed with sterilized water three times , after growth in 10% liquid V8 medium for 3 days , in order to produce zoospores . The zoospores were counted and adjusted to a concentration of 20 , 000/ml . 100 zoospores in 5 µl were inoculated onto etiolated hypocotyls of the susceptible soybean cultivar Hefeng 47 , which were grown at 25°C for 4 – 5 days at dark . After two days incubation at 25°C in the dark , the hypocotyls were photographed , and root segments were collected for biomass assays . For P . sojae infection assays on soybean hairy roots , soybean hairy roots expressing the constructs of interest were selected and placed on wet paper in a petri dish . The roots were inoculated with 10% V8 juice agar plugs containing freshly grown RFP-labeled P . sojae ( P6497-mRFP ) . After 48 − 72 hr incubation at 25°C , the number of oospores in one microscopic field was photographed and counted under a fluorescent microscope from 20 inoculated hairy roots . To quantitate the pathogen biomass in P . sojae infection sites , samples of 2 cm-length soybean hairy roots or hypocotyls from infection sites were collected and genomic DNA qPCR was used to confirm the relative biomass of P . sojae to soybean from a pool of 20 inoculated hairy roots or hypocotyls . The P . sojae actinA gene ( ID: 108986 ) was used to evaluate the P . sojae biomass by comparison to soybean gene CYP2 ( TC224926 ) . Total RNA was extracted from soybean hairy roots using a NucleoSpin RNA II kit ( Invitrogen ) according to the manufacturer’s instructions . To synthesize first strand cDNA , we used PrimeScript1 st Strand cDNA Synthesis Kit ( Takara , D6110A ) with 2 μg of RNA . Real-time qRT-PCR was performed in 20 μl reactions using Power SYBR Green ( Applied Biosystems ) following manufacturer’s instructions . GmCYP2 was used as the internal standard gene and the gene primers were listed at Supplementary file 1 . The reaction was conducted in an ABI Prism 7500 Fast Real-Time PCR System ( Applied Biosystems Inc . , Foster City , CA , USA ) . For each qPCR reaction , the reaction protocol was 95°C for 30 s followed by 40 cycles at 95°C for 5 s and 60°C for 34 s , followed by a dissociation step , 95°C for 15 s , 60°C for 1 min and 95°C for 15 s . Relative transcript levels were calculated using the 2-ΔΔCT method provided by the ABI 7500 System Sequence Detection Software . After 24 – 48 hr following A . tumefaciens inoculation , localization of fluorescently labeled proteins was observed using an LSM 710 laser scanning microscope with the following excitation wavelengths: GFP , 488 nm; and RFP , 561 nm ( Carl Zeiss , Jena , Germany ) . A . tumefaciens cells carrying recombinant constructs were infiltrated into N . benthamiana leaves after mixing with A . tumefaciens cells carrying the P19 silencing suppressor in a 1:1 ratio . 48–72 hr after infiltration , leaves were harvested and ground in cold lysis buffer ( 10 mM Tris-Cl ( pH 7 . 5 ) , 100 mM NaCl , 0 . 5 mM EDTA , 0 . 5% NP-40 ) containing 1 mM phenylmethylsulfonyl fluoride ( PMSF ) and 1 × protease inhibitor cocktail ( Sigma-Aldrich , St Louis , MO , USA ) . For immune-purification experiments , 72 hr after infiltration , the protein extracts were incubated with GFP-Trap_M beads ( Chromotek , ABIN509397 , Planegg-Martinsried , Germany ) for 3 hr at 4°C . After that , the beads were collected and washed three times in 700 μl of washing buffer ( 10 mM Tris-Cl ( pH 7 . 5 ) , 100 mM NaCl and 0 . 5 mM EDTA ) with 1 mM phenylmethylsulfonyl fluoride ( PMSF ) and 1 × protease inhibitor cocktail . For mass spectrometry analysis of proteins , the bound proteins were eluted by adding 50 μl of 0 . 2 M glycine ( pH = 2 . 5 ) for 30 s with constant mixing , followed by 5 μl of 1 M Tris base ( pH = 10 . 4 ) for neutralization . For mass spectrometry analysis , after centrifugation at 2500 g for 2 min , the supernatants were stored at −70°C . For other purposes , the proteins were dissociated from the washed beads by boiling for 5 min at 95°C in 20 µl washing buffer . The supernatants were then used for SDS-PAGE western blot assays . For in vitro pull-down assays , GST-tagged GmTAP proteins and His-tagged PsAvh52 protein were produced by expression of pEGX4T-2-GmTAP1 , pEGX4T-2-GmTAP2 and pET32a-PsAvh52 , respectively , in E . coli strain Rosetta 2 . The conditions for induction of protein expression were as follows: pEGX4T-2-GmTAP1 , GmTAP1M1 , GmTAP2 ( 16°C , 0 . 3 mM IPTG ) and pET32a-PsAvh52 ( 16°C , 0 . 1 mM IPTG ) for 16–20 hr . Total soluble proteins were extracted from harvested E . coli cells by ultrasonic cell disruption using lysis buffer ( 50 mM Tris , pH 8 . 0 , 50 mM NaCl , 1 × protease inhibitor mixture , 1 mM PMSF ) . Pull-down assays were performed as follows . The GST-GmTAP1 or GST-GmTAP2 proteins were incubated with 50 μl glutathione agarose beads ( GST beads ) for 3 hr at 4°C . After that , the GST beads were washed three times with lysis buffer and incubated with total soluble proteins from E . coli cells expressing His-PsAvh52 for 5–10 hr at 4°C . The beads were collected and washed three times in 700 μl of washing buffer ( 50 mM Tris , pH 8 . 0 , 50 mM NaCl , 1 × protease inhibitor mixture , 1 mM PMSF ) . The beads were then boiled for 5 min at 95°C in 20 µl washing buffer to dissociate the proteins from beads . The co-precipitation of His-PsAvh52 was detected by western blotting using anti-His antibody . Proteins were separated in SDS-PAGE gels and the separated proteins were transferred to PVDF membranes . The membranes were then blocked using 5% non-fat milk in PBST buffer ( 1 × PBS + 0 . 1% Tween 20 ) ( PBSTM ) for 30 min at room temperature with 60 r . p . m . shaking . The appropriate antibodies were then added to the PBSTM . Antibodies used were: anti-GST ( 1:5000; #M20007; Abmart ) , anti-His ( 1:5000; #M30111; Abmart ) , Anti-Flag ( 1:5000; #F3165; Sigma-Aldrich ) , anti-GFP ( 1:5000; #M20004; Abmart ) , and anti-RFP ( 1:5000; #5f8; Chromotek ) . After addition of the antibodies , the membranes were incubated at room temperature for 2–4 hr or 4°C for overnight; then washed three times ( 5 min each ) with PBST buffer . The membranes were then incubated with a goat anti-mouse ( Odyssey , no . 926 – 32210; Li-Cor ) or anti-rabbit ( Odyssey , no . 926–32211; Li-Cor ) IRDye 800CW antibody ( Odyssey , no . 926 – 32210; Li-Cor ) at a dilution of 1:10 , 000 in PBSTM at room temperature for 30 min with 60 r . p . m . shaking; and followed by three washes ( 5 min each ) with PBST . The signals were detected by excitation at 700 and 800 nm using a double color infrared laser imaging system ( Odyssey , LI-COR company ) . For the in vitro histone acetyltransferase ( HAT ) assays , GST-tagged GmTAP proteins and His-tagged PsAvh52 were expressed in E . coli and purified on GST- or NiNTA-beads as described above , following the manufacturer’s protocol . For the HAT assay , reaction mixtures of 30 μl contained 8 μg core chicken histones ( Millipore , 13 – 107 ) , 2 . 5 μg Acetyl-CoA ( Sigma-Aldrich , A2056 ) , 10 μg purified GmTAP1 or GmTAP1M1 proteins in HAT assay buffer ( 50 mM Tris-Cl pH 8 . 0 , 100 mM NaCl , 5 mM MgCl2 , 1 mM DTT , 5% glycerol ) . The reaction was incubated for 1 – 2 hr at 30°C . Then 7 µl 5 × SDS PAGE sample buffer was added to the mixture and heated at 95°C for 5 min . To test whether PsAvh52 affected the enzyme activity of GmTAP1 , His-PsAvh52 was incubated with GST-GmTAP1 at 4°C for 1 hr before initiating the enzyme assay . Western blots were performed to check the levels of histone acetylation using one of the following antibodies: anti-H3ac ( Millipore , 382158 ) ; anti-H3K9ac ( Millipore , 06 – 942 ) ; anti-H3K14ac ( Abcam , ab52946 ) ; anti-H2K5ac ( Abcam , ab45152 ) ; anti-H2AK9ac ( Abcam , ab177312 ) . The levels of histone acetylation were quantified by ImageJ software . For in vivo HAT assays , GFP , GmTAP1 , NLS-GmTAP1 , NES-GmTAP1 and NLS-GmTAP1M1 were expressed in N . benthamiana leaves as described above and then nuclear proteins were extracted as follows: ( 1 ) 6 g harvested plant tissue from each sample were ground in liquid nitrogen and then placed into a 50 ml BD tube with 30 ml TBST ( 1 × TBS + 0 . 1% tween 20 ) ; ( 2 ) the BD tubes with samples were shaken gently at 4°C for 15 min; ( 3 ) the samples were filtered into new BD tubes with 2 layers of micro-cloth and centrifuged at 1500 g for 8 min at 4°C; ( 4 ) the supernatants were removed and each precipitate was resuspended in 800 µl 50% sucrose; ( 5 ) the suspensions were gently added under 800 µl 25% sucrose in 2 ml tubes and centrifuged at 13000 g for 1 min at 4°C; ( 6 ) steps ( 4 ) and ( 5 ) were repeated 7 times; ( 7 ) the final nuclear precipitates were resuspended in 100–300 µl nuclear lysis buffer ( 10 mM Tris-HCl , 1 mM EDTA , pH 8 . 0 , 1% SDS , 1 mM phenylmethylsulfonyl fluoride , 1 × protease inhibitor cocktail ) and vigorously shaken at 4°C for 30 min; ( 8 ) the lysates were centrifuged at 13000 g for 1 min at 4°C , then the supernatants were transferred into new 2 ml tubes; ( 9 ) 5 × SDS loading buffer ( 1/5 total volume ) was added to the supernatants and the samples were boiled for 7 min . Then the samples were frozen until needed . To test the levels of acetylation of the nuclear core histones , samples were analyzed using western blotting after 15% SDS-PAGE with the antibodies listed above . Soybean hairy roots expressing PsAvh52-overexpression constructs or containing a control empty vector ( EV ) were inoculated with P . sojae and harvested 6 hr later . ChIP analysis was carried out as described ( Nagaki et al . , 2003; Wu et al . , 2011 ) . ChIP assays were performed after extracting chromatin . 5 g harvested plant tissue from each sample were ground in liquid nitrogen , washed twice using 10 ml M1 buffer ( 0 . 01 M Potassium Phosphate pH 7 . 0 , 0 . 1 M NaCl , β-mercaptoethanol , 11 . 85% Hexylene glycol ) then washed twice with 10 ml M2 buffer ( 0 . 01 M Potassium Phosphate pH 7 . 0 , 0 . 1 M NaCl , 0 . 01 M MgCl2 , β-mercaptoethanol , 11 . 85% Hexylene glycol , 0 . 5% Triton X-100 ) . 20 μl micrococcal nuclease ( MNase: NEB M0247S ) was added to digest nuclei in 2 ml MNB buffer ( 10% Sucrose , 0 . 05 M Tris-HCl , pH 7 . 5 , 4 mM MgCl2 , 1 mM CaCl2 ) for 10 min at 37°C . After then , 50 μl EDTA ( 0 . 5 M ) is added to stop the nuclease digestion before adding antibodies . 3 µg antibodies ( anti-H2K5ac , anti-H3K9ac ) were added to the chromatin solution and agitated at 4 ˚C overnight . 30 μl protein A Dynabeads ( Thermofisher , 10001D ) were added to the chromatin-antibody solution and agitated for 6 hr or overnight at 4 ˚C . The beads were recovered using magnetic separation then washed successively with 1 ml buffer A ( 50 mM Tris-HCl , pH 7 . 5 , 10 mM EDTA ) , containing 50 mM NaCl , 100 mM NaCl , or 150 mM NaCl . 400 μl elution buffer ( 20 mM Tris-HCl , pH 7 . 5 , 50 mM NaCl , 5 mM EDTA , 1% SDS ) was added twice to elute the DNA at 42°C for 15 mins . The two eluates were combined and extracted using phenol-chloroform isoamyl Alcohol ( 25:24:1 ) to remove remaining proteins , then the DNA was precipitated using ethanol overnight at −20°C , and washed twice with 70% ethanol . The sample was dried and dissolved in 20 μl ddH2O . As controls , input DNA was recovered by phenol extraction after the nuclease digestion step . As a second control , the antibody was omitted from the immunoprecipitation procedure . To quantitate the genes present in the DNAs purified from the precipitated chromatin , the appropriate primers were added to qPCR reactions with the respective DNAs , and qPCR was performed . Specific primers ( Supplementary file 1 ) corresponded to a short ( ~100 bp ) gene-specific region within 500 bp following the transcription start site of each gene . The ratio for each gene of interest was calculated from the chromatin samples from the roots expressing the constructs of interest compared to those containing the empty vector , after normalizing to the respective inputs . Each ratio was then divided by the same ratio calculated for the negative control gene ( UBQ5 ) . Transcript IDs of P . sojae genes in the JGI Physo3 database ( http://genome . jgi . doe . gov/Physo3/Physo3 . home . html ) : PsAvh52 ( ID: 356704 ) , PsAvh53 ( ID: 287363 ) , PsAvh137 ( ID: 288597 ) . Soybean genes in Phytozome database ( https://phytozome . jgi . doe . gov/pz/portal . html ) : GmTAP1 ( ID:Glyma . 18G216900 . 1 ) , GmTAP2 ( ID:Glyma . 09G272400 ) .
Oomycete pathogens secrete host cell-entering effector proteins to manipulate host immunity during infection . We previously showed that PsAvh52 , an early-induced RxLR effector secreted from the soybean root rot pathogen , Phytophthora sojae , could suppress plant immunity . Here , we found that PsAvh52 is required for full virulence on soybean and binds to a novel soybean transacetylase , GmTAP1 , in vivo and in vitro . PsAvh52 could cause GmTAP1 to relocate into the nucleus where GmTAP1 could acetylate histones H2A and H3 during early infection , thereby promoting susceptibility to P . sojae . In the absence of PsAvh52 , GmTAP1 remained confined to the cytoplasm and did not modify plant susceptibility . These results demonstrate that GmTAP1 is a susceptibility factor that is hijacked by PsAvh52 in order to promote epigenetic modifications that enhance the susceptibility of soybean to P . sojae infection .
Just like animals , plants can become infected and diseased . Among the microbes that infect plants , one group tends to stand out . Named Phytophthora after the Greek for ‘the plant destroyer’ , these fungus-like microbes cause diseases in many species of plant , including important food crops . These diseases are difficult to control , and as a result Phytophthora diseases cost the farming industry billions of dollars every year . Effective control of Phytophthora diseases is likely to depend on scientists first gaining a better understanding of how these microbes infect plants . Also like animals , plants have an immune system to protect themselves from disease . Yet many disease-causing microbes make so-called effector proteins to overcome their hosts’ defenses . Previously in 2015 , researchers reported that one effector made by a species known as Phytophthora sojae could suppress the immune system of soybean plants during the early stages of an infection . But it was not clear how the effector achieved this . Now , Li et al . , who include many of the researchers involved in the 2015 study , go on to show that the same effector , known as PsAvh52 , helps P . sojae to infect soybean plants by interacting with a previously unknown soybean enzyme . The enzyme is a transacetylase , meaning it belongs to a group of enzymes that transfer a chemical marker called an acetyl group on to other molecules including proteins . Li et al . went on to show that the PsAvh52 effector essentially hijacks the transacetylase enzyme , moving it to a location in the cell nucleus where it could chemically modify the proteins that package the soybean plant’s DNA . These chemical changes activate nearby genes that would have otherwise been switched off , and these incorrectly activated genes make the plant more susceptible to the infection . By deciphering one of the strategies that helps P . sojae to infect soybean plants , Li et al . have uncovered two possible approaches that may help to get this plant disease under control . The findings highlight the effector PsAvh52 as a weapon that could be blocked; they also reveal the transacetylase enzyme as a vulnerable point in the plant that could be protected . The next step will be to explore if there are chemical or genetic means that can achieve either of these two goals .