Two Trichome Birefringence-Like Proteins MediateXylan Acetylation, Which Is Essential for Leaf BlightResistance in Rice1[OPEN]

Yaping Gao2, Congwu He2, Dongmei Zhang, Xiangling Liu, Zuopeng Xu, Yanbao Tian, Xue-Hui Liu,Shanshan Zang, Markus Pauly, Yihua Zhou*, and Baocai Zhang*

State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academyof Sciences, Beijing 100101, China (Y.G., C.H., D.Z., X.L., Z.X., Y.T., Y.Z., B.Z.); University of Chinese Academyof Sciences, Beijing 100049, China (Y.G., D.Z., Y.Z.); Core Facility for Protein Research, Institute of Biophysics,Chinese Academy of Sciences, Beijing 100101, China (X-H.L, S.Z.); and Institute for Plant Cell Biology andBiotechnology, Heinrich-Heine University, 40225 Düsseldorf, Germany (M.P.)

ORCID IDs: 0000-0001-5202-4205 (Y.G.); 0000-0003-4560-9058 (D.Z.); 0000-0001-9052-6185 (Z.X.); 0000-0002-2988-7296 (S.Z.);0000-0001-6644-610X (Y.Z.); 0000-0002-3239-7263 (B.Z.).

Acetylation is a ubiquitous modification on cell wall polymers, which play a structural role in plant growth and stress defenses.However, the mechanisms for how crop plants accomplish cell wall polymer O-acetylation are largely unknown. Here, we report onthe isolation and characterization of two trichome birefringence-like (tbl) mutants in rice (Oryza sativa), which are affected in xylanO-acetylation. ostbl1 and ostbl2 single mutant and the tbl1 tbl2 double mutant displayed a stunted growth phenotype with varieddegree of dwarfism. As shown by chemical assays, the wall acetylation level is affected in the mutants and the knock-down andoverexpression transgenic plants. Furthermore, NMR spectroscopy analyses showed that all those mutants have varied decreases inxylan monoacetylation. The divergent expression levels ofOsTBL1 andOsTBL2 explained the chemotype difference and indicated thatOsTBL1 is a functionally dominant gene. OsTBL1 was found to be Golgi-localized. The recombinant OsTBL1 protein incorporatesacetyl groups onto xylan. By using xylopentaose, a preferred acceptor substrate, OsTBL1 can transfer up to four acetyl residues ontoxylopentaose, and this activity showed saturable kinetics. 2D-NMR spectroscopy showed that OsTBL1 transfers acetate to both 2-Oand 3-O sites of xylosyl residues. In addition, ostbl1 and tbl1 tbl2 displayed susceptibility to rice blight disease, indicating that this xylanmodification is required for pathogen resistance. This study identifies the major genes responsible for xylan acetylation in rice plants.

The plant cell wall, a sophisticated multiple polymernetwork, encases plant cells and plays essential roles inplant growth and development, including morphogene-sis, water and nutrient transduction, and protection of

plants against abiotic and biotic stresses. To achieve thesefunctions, plants often incorporate acetyl substituents intocell wall polymers (Gille and Pauly, 2012). Hence, manycell wall polymers, including pectin, several kinds ofhemicellulose, and lignin, are O-acetylated (Gille andPauly, 2012; Pawar et al., 2013). As O-acetylation impactsphysicochemical property of polymers, the abundanceand position of acetyl groups on the polymers vary indifferent plant species and tissues and across various de-velopmental stages. Acetyl substitution on xyloglucan(XyG), the major hemicellulose in the dicot primary wall,often occurs on side chains, specifically the galactosylresidues atO-3,O-4, orO-6 (Gille et al., 2011b). Only grassanddicotyledonous Solanaceae have anO-acetylatedXyGbackbone (Schultink et al., 2014; Liu et al., 2016). The acetylmoieties on the backbone glucosyl residues are at O-6(Hoffman et al., 2005; Jia et al., 2005). Xylan is the mostabundant hemicellulose in nature and has a highlyacetylated backbone. The degrees of acetylation rangefrom 0.3 to 0.6 acetate to Xyl ratio depending on the plantspecies (Teleman et al., 2002; Yuan et al., 2016c). Acetylgroups are attached to xylan xylopyranosyl residues atO-2 orO-3 andO-3 ifO-2 contains other substituents, forexample, GlcA/MeGlcA/Araf (Teleman et al., 2000). Inaddition to these two polymers, glucomannans, pectins,

1 This study was supported by the National Natural Science Foun-dation of China (31530051), the Ministry of Sciences and Technologyof China (2013CB127001, 2012CB114501), the Ministry of Agricultureof China for Transgenic Research (grant 2016ZX08009-003-003), andYouth Innovation Promotion Association CAS (2016094).

2 These authors contributed equally to the article.* Address correspondence to bczhang@genetics.ac.cn or yhzhou@

genetics.ac.cn.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Baocai Zhang (bczhang@genetics.ac.cn).

Y.G., B.Z., and Y.Z. conceived the project and research plans; Y.G.and C.H. performed mutant identification; Y.G., C.H., and B.Z. per-formed biochemical experiments and data analyses; D.Z. and Y.T.performed subcellular localization experiments; X.L. and Z.P. per-formed transformation and field works. X.-H.L., S.Z., and M.P. pro-vided technical assistance in NMR experiments; M.P. reviewed andedited the article; B.Z. and Y.Z. wrote the article.

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and lignin were also found to be O-acetylated at certainpositions (Liners et al., 1994; Schols and Voragen, 1994;Lundqvist et al., 2002; del Río et al., 2008; Gille et al.,2011a). Although monoacetylation is the major modifi-cation form, some polymers, such as XyG, xylan, andpectins, can be substituted by two O-acetyl-groups onone sugar residue. However, the molecular mechanismfor how plants process and regulate cell wall polymerO-acetylation is not well understood.Recent progress indicates that at least three groups of

proteins, RWAs (Reduced Wall Acetylation), TrichomeBirefringence-Like (TBL), and AXY9 (Altered Xyloglucan9), are involved in the polymer O-acetylation. RWAsconsist of 10 transmembrane helices homologous to theCnCas1p from Cryptococcus (Manabe et al., 2011). Arabi-dopsis (Arabidopsis thaliana) possesses four RWA homo-logs in its genome, and mutation of RWA2 results inan overall reduction of acetylation on several polymers(Manabe et al., 2011). The quadruple mutant of all fourRWA genes exhibited similar butmore severe defects (Leeet al., 2011), indicating that RWAs might function astransporters by supplying acetyl-donor(s) for polysac-charide O-acetylation into the Golgi apparatus (Gille andPauly, 2012). AXY9 is a recently reported Golgi-localizedprotein bearing only one transmembrane domain. Thedecrease in acetylation level onmultiple polymers such asXyG and xylan in the axy9 mutant indicates its involve-ment in O-acetylation, but its precise function remainselusive (Schultink et al., 2015). TBL proteins share twoconserved domains, the TBL and DUF231 domain, withthe Arabidopsis Trichome Birefringence protein (Potikhaand Delmer, 1995; Bischoff et al., 2010). Each domainharbors one conservedmotif, aGly-Asp-Ser (GDS) and anAsp-x-x-His (DxxH), respectively (Gille and Pauly, 2012).Based on the sequence similarity, 46 TBL membershave been found in Arabidopsis, potentially differingin their polymer specificity. Mutations in AXY4/TBL27and AXY4L/TBL22 lead to compromised acetyl-XyG,providing genetic evidence for this hypothesis (Gilleet al., 2011b). Furthermore, ESK1/TBL29 was identifiedas a xylan acetyltransferase, because the esk1 mutant dis-plays a specific defect in 2-O- and 3-O-monoacetylation onxylan (Xiong et al., 2013; Yuan et al., 2013). SeveralESK1 close homologs, TBL3, TBL31, TBL32, TBL33,TBL34, and TBL35, have been characterized to be addi-tional candidates for xylan acetylation, probably withvaried activity or regiospecificity (Yuan et al., 2016a,2016b, 2016c). Nevertheless, ESK1 is so far the only TBLbeing biochemically documented to be able to transfer theO-acetyl substituent to the 2-O and 3-O positions ofxylopyranosyl residues in vitro (Urbanowicz et al., 2014).Biochemical activities of the other TBL proteins remainunknown.The wide existence of acetylation modification on

wall polymers indicates functional importance. How-ever, the impetus that promotes plants to evolve thisspecific modification is not clear. One possibility is thatthis modification can enhance the interaction of thehemicelluloses with other wall polymers (Busse-Wicheret al., 2014), leading to the rigid wall conformation to

facilitate relevant physiological functions. Several Arabi-dopsis mutants that have the compromised acetyl-xylanexhibit collapsed xylem vessels and abnormal growthstatus (Lee et al., 2011; Xiong et al., 2013; Schultink et al.,2015; Yuan et al., 2016c). Another potential function ofpolymer O-acetylation is to protect plants against invad-ing microorganism and environmental stresses. The esk1(tbl29) mutant displays freezing resistance in the absenceof cold acclimation (Xin and Browse, 1998). pmr5 (tbl44)exhibits tolerance to powderymildew (Vogel et al., 2004).axy4 (tbl27) mutant was more sensitive to aluminumtreatments (Zhu et al., 2014). These data suggest thatacetylation of wall polymers is vital for plant growth andadaptation to various environments.

Rice (Oryza sativa) is one of the world’s most impor-tant crops. With its grass-specific wall structure, rice isused as a model for understanding cell wall biosyn-thesis in monocots. The rice genome contains more TBLmembers than Arabidopsis. But none of rice TBL mem-bers have been characterized. It is therefore unclear whatagronomic trait is correlated with polysaccharide acety-lation. Here, we report on the functional characterizationof two rice TBL proteins, OsTBL1 andOsTBL2, which arehomologs of Arabidopsis TBL34 (Yuan et al., 2016b). Weprovide several lines of biochemical and genetic evidenceto demonstrate that OsTBL1 and OsTBL2 are xylan ace-tyltransferases, which is responsible for xylan backbonemonoacetylation and resistance to bacterial leaf blightdisease.

RESULTS

Phylogenetic Analysis of TBL Family in Rice

Blastp and domain searches in the rice genome database(RiceGenomeAnnotationProject, http://rice.plantbiology.msu.edu/) were performed to systematically identify TBLmembers in the rice genome. As a result, 66 TBLmemberswere found distributed over 10 chromosomes, with thehighest density on chromosome 6, where 20 TBL genesare clustered. The rice TBL proteins are composed of aconserved TBL domain and a DUF231 domain with anaverage length of 457 amino acids, except Os04g58340,Os06g15560, and Os06g15580, which do not containa DUF231 domain (Supplemental Table S1). Gene ex-pression analysis showed that 61 TBLs have differentexpression profiles (Supplemental Table S2), indicatingthat they may function in a spatio-temporal manner.Based on the sequence similarity of 46 Arabidopsis and66 rice TBLs, we constructed a phylogenetic tree usingMaximum likelihood. The Arabidopsis TBLs that havebeen identified to affect XyG and xylan acetylation wereclustered into different clades (Fig. 1; Gille et al., 2011b;Xiong et al., 2013; Yuan et al., 2016a, 2016b, 2016c). Inaddition, most Arabidopsis TBL proteins have at leastone rice homolog (Fig. 1), indicating that gene expansionevents seem to occur after themonocot-dicot divergence.Arabidopsis TBL29 is the only TBL biochemically iden-tified as a xylan acetyltransferase (Urbanowicz et al.,2014). Rice has 15 TBL29 homologs. We sequentially

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named the 66 rice TBL proteins from this clade, fromOsTBL1 to OsTBL66 (Fig. 1).

Mutations in OsTBL1 and OsTBL2 Disrupt MonoO-Acetylation of Xylan

To address the function of these rice TBL genes, weisolated two Tos17 insertional mutants in Os12g01560/OsTBL1 andOs11g01570/OsTBL2 (Fig. 2, A and B). Bothostbl1 and ostbl2 mutants exhibited a stunted growthhabit, and ostbl1 looks severe compared to ostbl2 (Fig. 2,C and D). Sequence alignment revealed that both TBLsharbor the conserved TBL and DUF231 domains withup to 97% sequence identity (Supplemental Fig. S1).The double mutant, tbl1 tbl2, showed reduced plantheight similar as ostbl1 (Fig. 2D). In addition, we gen-erated OsTBL1 and OsTBL2 knock-down plants by

artificialmiRNAapproach and the overexpression plants.The knock-down plants mimicked the mutant pheno-types, showing the reduced plant height, whereas theoverexpression lines were morphologically indistin-guishable from the wild-type plants, although quan-titative real-time PCR (qRT-PCR) revealed an increasedexpression level of OsTBL1 and OsTBL2 in the corre-sponding lines (Supplemental Fig. S2).

Next, we examined structural wall attributes of wildtype, mutants, and the transgenic lines. No significantreduction in neutral monosaccharide and cellulosecontent was found in the mutants and transgenic lines(Supplemental Table S3). We further examined the ac-etates bound on cell wall polymers. ostbl1 and ostbl2contained 16% and 10% fewer acetyl esters compared towild type, respectively, similar to decreases observed inthe knock-down plants (Fig. 3A). The acetate level in the

Figure 1. Phylogenetic analysis of the TBL super family in rice and Arabidopsis. The tree was built by MEGA6 with bootstrapsupport (1,000 replicates). The red stars indicate the reported Arabidopsis TBLs. The arrow indicates OsTBL1 and OsTBL2.

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double mutants was decreased by 20%. Although in theOsTBL1 overexpression plants an increased acetyl-contentcould be observed (Fig. 3A), no acetate alteration wasdetected in the OsTBL2 overexpression plants, which

might be due to the low expression level ofOsTBL2. Itcould be concluded that OsTBL1 and OsTBL2 areinvolved in wall O-acetylation. To identify the wallpolymer(s) that were affected, deuterated dimethyl

Figure 2. Phenotypic analysis of ostbl1,ostbl2, and tbl1 tbl2 double mutants. A,The Tos17 insertion position in OsTBL1and OsTBL2. B, Confirmation of the Tos17insertion in the mutants by PCR with theprimers shown in (Supplemental Table S4).C, The mature plants of ostbl1, ostbl2, andtbl1 tbl2 double mutants. D, Measurementof plant height of the matured wild-typeand mutant plants. Error bars indicate SDfrom the mean height of 16 plants of eachgenotype. **P , 0.01 by Student’s t test.Bars = 10 cm.

Figure 3. ostbl1 and ostbl2 mutants showdefects in xylanO-acetylation. A, Analysesof the acetate content in wall residues ofthe internodes from wild type, mutants,and knock-down (kd) and overexpression(ox) plants. B, 2D-HSQC NMR spectra ofcell wall residues of the internodes fromwild type, ostbl1, ostbl2, and the tbl1 tbl2double mutants. C, Quantification of theacetyl signals on xylan shown in B. D,Analyses of acetyl ester content on acetyl-xylans extracted from wild type, ostbl1,ostbl2, and tbl1 tbl2 double mutants. Errorbars in this figure indicate SD from themean of three replicates. *P , 0.05 and**P , 0.01 by Student’s t test.

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sulfoxide (DMSO-d6) dissolved cell wall residueswere subjected to NMR spectroscopy. Heteronuclearsingle quantum coherence (HSQC) analyses showedthat the signal intensity of 3-O-Ac-b-D-Xylp was sig-nificantly decreased in the mutants, whereas 2-O-Ac-b-D-Xylp was slightly altered (Fig. 3, B and C). Toverify this finding, acetyl-xylans were extracted fromthe wild type and mutants by DMSO and their acetylester content was examined. The acetate content wasdecreased by 47% and 12% in ostbl1 and ostbl2, re-spectively, while a 55% reduction was found in thetbl1 tbl2 double mutant (Fig. 3D). Therefore, OsTBL1and OsTBL2 are required for xylan monoacetylation.

In Arabidopsis, defects in xylan acetylation often causecollapsed vessels (Xiong et al., 2013; Yuan et al., 2013). Wetherefore examined the anatomic structure of internodesin wild type, ostbl1, ostbl2 and the double mutants. Noobviously morphological changes were detected in xylemvessels (Supplemental Fig. S3). Probably due to the di-vergent pattern of vascular system between the monocotand dicot species, the decreased acetylation level resultedfrom OsTBL1 and OsTBL2 mutations does not cause col-lapsed vessels in rice.

OsTBL1 Has a Major Effect Compared to OsTBL2 in Rice

The above analyses displayed varied abnormalitiesin ostbl1 and ostbl2, in which ostbl1 looks more severethan ostbl2. We proposed that such phenotypic diver-gence may result from gene expression rather thanbiochemical activity, because their protein sequencesare almost identical. To address this possibility, wecompared the expression levels of OsTBL1 and OsTBL2in the wild-type tissues. The transcript levels ofOsTBL1are indeedmuch higher than those ofOsTBL2 in the riceinternodes as revealed by qRT-PCR (Fig. 4A). More-over, we examined whether there was a change inexpression of OsTBL1 in a ostbl2 line and vice versa.qRT-PCR analyses showed that the expression level ofOsTBL1 is unchanged in ostbl2, whereas OsTBL2 isup-regulated by 1.5-fold in ostbl1 (Fig. 4B). Consider-ing that overexpression of OsTBL2 up to 5-fold did notshow a phenotype (Fig. 3; Supplemental Fig. S2), thephenotypes observed in both mutants are mainly attrib-uted to the gene mutation itself, respectively. To verifythis conclusion at a protein level, polyclonal antibodiesagainst OsTBL1/2 were generated. Western blottingdetected only one band in the wild type and an identicalbut faint and wild-type-like band in ostbl1 and ostbl2,respectively (Fig. 4C). No signal was observed in thedouble mutant (Fig. 4C). Therefore, although this anti-body was specific for OsTBL1 and OsTBL2, it could notdistinguish between the two isoforms. The stronger sig-nals observed in the ostbl2mutant are in agreement withthe higher levels of transcripts observed for OsTBL1,suggesting that the phenotypic deviations in ostbl1 andostbl2 mutants are likely due to varied transcript andprotein abundance in the mutants. Taken together,OsTBL1 has the dominant phenotypic effect.

OsTBL1 Is a Golgi-Localized Protein

As OsTBL1 and OsTBL2 share almost identical pro-tein sequence, the following study was focused onOsTBL1. The Golgi apparatus is a major organelle formatrix polysaccharide synthesis and acetylation (Gilleand Pauly, 2012). To determine whether OsTBL1 isGolgi-localized, we fused OsTBL1 with a green fluo-rescence protein (GFP) and cotransfected this chimericconstruct inNicotiana benthamiana leaves with a mCherry-fused Golgi marker, Man49. The overlapping signals in-dicated that OsTBL1 is localized in the Golgi apparatus(Fig. 5, A and B). To verify this finding in planta, weperformed Suc density gradient centrifugation and west-ern blotting of fractions prepared from wild-type seed-lings. The fractions labeled by anti-OsTBL1/2 antibodieswere largely identified by anti-IRX14 antibodies thatrecognize IRX14, a Golgi-protein involved in xylan bio-synthesis in rice (Chiniquy et al., 2013; Lee et al., 2014),suggesting that OsTBL1 and IRX14 are present in thesame organelle (Fig. 5C). Anti-BiP and anti-PIP1s anti-bodies that target to the endoplasmic reticulum andplasma membrane-containing fractions, respectively,were used as the control to monitor the Suc densitygradient fractionation (Fig. 5C). Moreover, to visualizesuch localization in the Golgi apparatus of rice cells, weperformed immuno-electron microscopy in root tips.The gold particles labeled by anti-OsTBL1 antibodies

Figure 4. Examination of OsTBL1 and OsTBL2 expression level. A, qRT-PCR analysis of OsTBL1 and OsTBL2 expression in the wild-type inter-nodes. Rice HNR was used as an internal control. Mean 6 SD of threereplicates. B, qRT-PCR analyses ofOsTBL1 andOsTBL2 in the internodes ofostbl2 andostbl1, respectively. Error bars indicate SDof triplicate replicates.RiceHNR andZCF61were used as internal controls for amplifyingOsTBL1and OsTBL2, respectively. C, Western blotting to examine OsTBL2 andOsTBL1 inostbl1,ostbl2, and the tbl1 tbl2doublemutant. BiPwas added tovisualize the loading amount in each lane.

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were present in Golgi stacks (Fig. 5D). Although anti-OsTBL1/2 cannot distinguish between OsTBL1 andOsTBL2, given that OsTBL1 is the more abundantlyexpressedgene, the signals generated by this antibodyweremainly derived from OsTBL1 (Fig. 4C). Therefore, weconcluded that OsTBL1 is localized in the Golgi apparatus.

OsTBL1 Exhibits O-Acetyltransferase Activity on theXylan Backbone

The biochemical activity of OsTBL1 was examined byexpressing the protein in Pichia pastoris (Supplemental Fig.S4). The recombinant proteins were purified and incubatedwith several commercial polysaccharides, including xylan,xyloglucan, pectin, and lignin, in an attempt to determineits acceptor substrate.Acetyl groupswere incorporated intoxylan 5-fold higher than that into other acceptor substrateswhen using 3H-labeled acetyl CoA as a donor (Fig. 6A),suggesting that xylan or xylooligomers is the native ac-ceptor substrate of OsTBL1. These biochemical data are inagreementwith the observed acetate defects in themutants.To determine what kind of xylooligomers OsTBL1 prefers,Xyl to xylohexaose (X6)were used as the acceptor substratefor enzyme activity assay. OsTBL1 could incorporate acetylsubstituents onto xylotriose to X6 with the highest incor-poration in xylopentaose (X5; Fig. 6B). Using X5 as the ac-ceptor substrate we analyzed the reaction products bymatrix-assisted laser-desorption ionization time of flight(MALDI-TOF) mass spectroscopy (MS). MALDI-TOFspectra revealed that OsTBL1 can transfer up to fouracetyl residues onto the X5 (Fig. 6, C and D).

Moreover, the amount of substituent on X5 reached amaximum (approximately 42%) while the reaction timewas increased up to 40 h (Fig. 7A). Considering that morethan 30% of X5 was acetylated by incubating with OsTBL1for 16 h (Fig. 7B), we examined Km and Vmax of OsTBL1under this condition. As shown in Figure 7C, the acetyl-transferase activity onto X5 showed saturable kinetics, witha Km of 4.98 mM and Vmax of 0.99 mM/min, respectively.Therefore, OsTBL1 is likely a xylan acetyltransferase.

To investigate the acetylation pattern on X5, the re-action products were further subjected to MALDI-TOF/TOF tandem MS analysis. Through analysis of thegenerated fragment ions of monoacetylated X5, the acetylgroup should locate on either internal or terminal xylosylresidues (Fig. 8A). We also detected diacetylated xylobi-ose and triacetylated xylotriose from the fragment ions oftriacetylated X5 (Fig. 8A). As diacetylation rarely occurson Xyl residue in rice wall, it is assumed that the acetylgroups can be transferred onto consecutive Xyl resi-dues by OsTBL1. To determine the regiospecificity ofOsTBL1 activity, we analyzed the OsTBL1 products by2D 1H-1H COSY spectroscopy. Signals correspondingto 3-O-Ac-b-D-Xylp and 2-O-Ac-b-D-Xylp, but not 3-O,2-O-di-Ac-b-D-Xylp, were observed (Fig. 8B), demonstrat-ing that OsTBL1 is a xylan monoO-acetyltransferase.

ostbl1 and ostbl2 Mutants Are Susceptible toBlight Disease

Polysaccharide O-acetylation can impact the abilityof plants resistant to biotic and abiotic stresses (Diener

Figure 5. OsTBL1 locates in the Golgi apparatus. A, Transient subcellular localization assay. OsTBL1 was fused with GFP andtransfected into tobacco leaves together withMan49-mCherry. B, Intensity plot of OsTBL1-GFPandMan49-mCherry shown in A,indicating that OsTBL1 and Man49 presence overlaps. C, Western blotting the fractions from Suc density gradient centrifugationwith the indicated antibodies. Abs, antibodies. D, Immunogold labeling of OsTBL1 in root tips with anti-OsTBL1/2 polyclonalantibodies. Arrows indicate particles in the Golgi (G) stacks. Bars = 10 mm (A) and 200 nm (D).

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and Ausubel, 2005; Xin et al., 2007; Manabe et al., 2011).Online expression data suggested that OsTBL1 iscoexpressed with multiple disease resistance response

genes such as LOC_Os03g28190/Os03g0400200 (Buellet al., 2005; Supplemental Fig. S5), indicating thatOsTBL1 may be induced by pathogens. Leaf blight is aworldwide rice disease and causes devastative yieldlosses (Zhang and Wang, 2013; Liu et al., 2014). To ex-amine whether OsTBL1 and OsTBL2 contribute to riceleaf blight disease resistance, two blight pathogenstrains, Pxo145 and Pxo61, were inoculated onto theleaves of wild-type and mutant plants. The lesionlength in the leaves of ostbl1 and tbl1 tbl2 was 2- and3-fold longer than those of wild-type plants, whereasthe lesion length in ostbl2 remained identical to thatof the wild type (Fig. 9). Therefore, OsTBL1 is essentialfor the resistance to leaf blight disease. Mutation inOsTBL1 alters the wall structure that may cause sus-ceptibility to this disease.

DISCUSSION

The Rice Genome Harbors More TBL MembersThan Arabidopsis

O-acetyl substitution is a common modification of cellwall polymers, which is important for polysaccharidenetwork stability and recalcitrance. Three groups of pro-teins have been identified to be involved inO-acetylationof cell wall polymers (Gille et al., 2011b; Manabe et al.,2011; Schultink et al., 2015), in which TBLs constitute thelargest gene family. Forty-six TBLmembers are present inthe Arabidopsis genome (Gille and Pauly, 2012), butonly a few of those TBLs have been characterized(Gille et al., 2011b; Xiong et al., 2013; Yuan et al., 2013,2016a, 2016c). Sequence analyses identified 66 TBLmembers in rice (Fig. 1). We therefore proposed thatrice may share a conservedmechanism in wall polymer

Figure 7. Analysis of OsTBL1 enzymatic property. A,Examination of acetyl incorporation activity ofOsTBL1 onto X5 in different reaction time (h). B,Quantification of the relative product abundance indifferent reaction time (h) as shown in A. Error barsindicate SD from the mean of three replicates. C,Enzyme kinetics of OsTBL1 determined bymeasuringthe acetic acid transferal to increasing amounts ofacceptor X5 (0–48mM) in 16-h reaction time. Km andVmax were calculated by the Michaelis-Menten plotsof three independent replicates.

Figure 6. OsTBL1 represents a xylanO-acetyltransferase. A, Examination ofacetyl incorporationactivityofOsTBL1onto the indicatedwall polymersusingradiolabeled acetyl-CoA as the donor. B, Analysis of acetyl incorporation byOsTBL1 onto Xyl (X1) up to X6 by LC-QTOF-MS. C, MALDI-TOF spectra ofthe reaction products of OsTBL1 by using X5 as the acceptor. The inlet showsthe magnified graph of the X5Ac4 peak m/z 869. D, Quantification of therelative product abundance shown in C. Error bars in this figure indicate SDfrom the mean of three replicates. **P, 0.01 by Student’s t test.

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O-acetylation. However, to date none of the rice TBLshave been functionally characterized. Here, throughisolation and characterization of ostbl1 and ostbl2 mu-tants, we found that mutations in both TBLs result in adecrease in xylan monoacetylation (Fig. 3), providingthe first evidence to our knowledge that rice TBLs areinvolved in cell wall acetylation. ostbl1 and ostbl2 mu-tants exhibited minor but different alterations in plantheight and acetyl-xylan content (Figs. 2 and 3). Ex-pression analyses at transcriptional and translationallevels in wild type and mutants revealed that OsTBL1seems to play a dominant role in xylanO-acetylation, asit is more abundant thanOsTBL2 (Fig. 4). However, theminor phenotypes shown in both TBL mutants sug-gested that additional TBLs may participate in xylanacetylation in rice. OsTBL3 is another gene clustered inthe same subclade of OsTBL1 and OsTBL2 (Fig. 1).Online microarray data and qRT-PCR analysis showedthat the expression level of OsTBL3 in many tissues ismuch lower than OsTBL1 (Supplemental Fig. S6).Within this clade, an additional 12 TBLs are present and

may possess similar enzyme activity. Further studiesthat investigate the biochemical specificity and ex-pression patterns of other TBL family members couldprovide evidence to explain the minor phenotypescaused by OsTBL1 and OsTBL2 mutations. Overall, theexistence of more TBL members in rice genome indi-cates the importance of wall O-acetylation in this crop.

OsTBL1 Represents a Xylan O-Acetyltransferase

To date, only TBL29 has been shown to have xylanO-acetyltransferase activity (Urbanowicz et al., 2014).Several TBL29 homologs were also identified to func-tion in xylan O-acetylation based on the characteriza-tion of mutant chemotypes (Yuan et al., 2016a, 2016b,2016c). The members clustered in this clade are likely tohave a similar function with varied activity or regio-specificity. Therefore, identifying the enzyme activity ofTBLs is crucial for understanding their functions. Fif-teen rice TBLs are grouped in this clade, and OsTBL1and OsTBL2 are orthologs of Arabidopsis TBL34 andTBL 35 (Fig. 1), which were shown to affect xylan acet-ylation without enzymatic characterization (Yuan et al.,2016b). OsTBL1 and OsTBL2 have canonical TBL struc-tures, including a TM domain at the N terminus and aTBL domain and a DUF231 domain containing the GDSand DxxH motifs, respectively (Bischoff et al., 2010; Gilleand Pauly, 2012; Supplemental Fig. S1). Here, we providemultiple lines of biochemical and genetic evidence tocorroborate that OsTBL1 is a xylan O-acetyltransferase.

Figure 8. OsTBL1 mediates xylan monoO-acetylation. A, MALDI- MS/MS spectra of monoacetylated X5 (m/z 743.20 [M+Na]+, top) and tri-acetylated X5 (m/z 827.23 [M+Na]+, bottom) generated by incubatingwith OsTBL1. B, COSY spectra of acetylated X5 in the absence (Mock)and the presence of OsTBL1. The COSYexperiment was carried out fortwo replicates.

Figure 9. OsTBL1 and OsTBL2 affect defense against leaf blight dis-ease. A to C, The phenotypes of leaves from wild types, ostbl1, ostbl2,and tbl1 tbl2 inoculated with water (Control, A) or the pathogen strainsPxo61 (B) and Pxo145 (C). The lesion lengths were measured after2-week inoculation. D, Statistics analysis of lesion length shown in Ato C. Bars = 1 cm. Error bars indicate SD of the mean of n $ 5 leaves.*P , 0.01 by Student’s t test.

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Firstly, OsTBL1 is located in the Golgi apparatus (Fig. 5).Secondly, the purified recombinant OsTBL1 displayedacetyl transferase activity on xylan in vitro (Fig. 6).Thirdly, when using acetyl-CoA as the donor, OsTBL1transferred up to four acetyl residues onto X5 and thisactivity showed saturable kinetics (Fig. 7). Fourthly, COSYanalysis showed thatOsTBL1 can transfer acetyl groups tothe 2-O and 3-O position of xylosyl residues, resulting inmonoO-acetylated xylooligosaccharides (Fig. 8). Mostimportantly, all of these biochemical findings were cor-roborated by the corresponding mutant analyses, inwhich the ostbl1, ostbl2, anddoublemutants had anoverallreduction in 2-O and 3-O monoacetylation of xylan (Fig.3). Since spontaneous migration of acetyl residues occursbetween different positions of a glycosyl-residue in vitroand in vivo (Gille and Pauly, 2012; Yuan et al., 2016a,2016c), the observed acetylation in plantsmight not reflectthe direct effect of enzyme activity. This might be thereason why the observed decrease in 2-Omonoacetylatedxylosyl residues is not as significant as at the 3-O positionin ostbl1 and ostbl2 and the double mutants (Fig. 3), whichis similar to the results found for the Arabidopsis tbl3 tbl31mutants (Yuan et al., 2016c). For this reason, we cannotconclude that OsTBL1 is specifically required for xylan2-O or 3-O monoacetylation, unless real-time acetatetransfer monitoring is implemented. Moreover, MALDI-TOF/TOF tandem MS analysis revealed that OsTBL1has the ability to add acetyl residues to consecutive Xylresidues (Fig. 8A), which has not been reported in Ara-bidopsis. There, xylosyl residues were found to be al-ternatively substituted with acetates (Busse-Wicher et al.,2014). OsTBL2 shares 97% sequence identitywithOsTBL1(Supplemental Fig. S1), and mutation in this proteincaused similar cell wall defects (Fig. 3). OsTBL2 is alsolikely a xylan O-acetyltransferase as OsTBL1.

The Functions of O-Acetylated Xylan in Rice

Xylan is a major hemicellulose in plant cell wall andO-acetylation is one of the most important modifica-tions on this polymer. The biological functions ofO-acetyl-xylan are still under investigation. The phe-notypes of several tbl mutants and transgenic plants in-clude collapsed xylem vessels, stunted growth, pathogendefense, and freezing tolerance, providing genetic cluesfor the roles ofO-acetyl-substituents in plant growth anddevelopment (Vogel et al., 2004; Xin et al., 2007; Lee et al.,2011; Manabe et al., 2011; Xiong et al., 2013; Yuan et al.,2016c). However, the function of O-acetylation of wallpolymers in crops is still unclear. Rice ostbl1 and ostbl2and the tbl1 tbl2 double mutants that have reduced levelof acetyl-xylan exhibit reduced growth phenotypes sim-ilar to those observed in Arabidopsis (Fig. 2). In addition,thesemutants showed susceptibility to leaf blight (Fig. 9),a devastating disease in rice production.

O-acetylation of wall polymers also represents a majorobstacle to the economic conversion of lignocellulosics tobiofuel (Loqué et al., 2015). Here, we examined the sac-charification efficiency of lignocellulosic material derived

from ostbl1, ostbl2, and the tbl1 tbl2 double mutants. Thesugar yields were not different between the mutants andthe wild type (Supplemental Fig. S7), suggesting that a10% to 20% acetate decrease in lignocellulosic material ofthese mutants is not sufficient for an improvement forsaccharification conversion rate in rice.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The rice (Oryza sativa) ostbl1 and ostbl2 mutants were ordered from the RiceTos17 Insertion Mutant consortium (http://tos.nias.affrc.go.jp). The Tos17 in-sertion sites were confirmed by sequencing (Supplemental Table S1). The tbl1tbl2 double mutant was generated by crossing ostbl1 and ostbl2 single mutantsduring the growing season. To generate the overexpression transgenic plants,the full coding sequences of OsTBL1 and OsTBL2 were amplified and insertedinto the pCAMBIA1300 vector between the CaMV 35S promoter and NOSterminator. We also generated the OsTBL1 and OsTBL2 knock-down plantsby artificial miRNA approach. The targeted fragments were amplified(Supplemental Table S4) and cloned into the pCAMBIA1300 vector containingOsa-miRNA528. The resulting constructs were transformed into Nipponbare, awild-type variety. The wild type, mutants, and transgenic plants used in thisresearch were grown in the experimental fields at the Institute of Genetics andDevelopmental Biology, Chinese Academy of Sciences (Beijing, China) and atLinshui, Hainan Province, during the natural growing season.

Bioinformatic Analyses

Rice TBLs were identified based on the annotation of the rice genome da-tabase (Rice Genome Annotation Project, http://rice.plantbiology.msu.edu/).An unrooted tree of the TBLs in rice and Arabidopsis (Arabidopsis thaliana) wasgenerated using Maximum Likelihood with the MEGA6 software (Tamuraet al., 2013) with 1,000 bootstrap replications. The spatio-temporal expressionprofiles of OsTBL1 and OsTBL3were analyzed based on the expression data inRiceXPro database (http://ricexpro.dna.affrc.go.jp/). Alignment of OsTBL1and OsTBL2 proteins was performed using ClustalX. Coexpression analysis ofOsTBL1was conducted by using the online data from the Rice FREND database(http://ricefrend.dna.affrc.go.jp/).

Chemical Assays

The second internodes from mature wild type, mutants, and transgenicplants were collected for the preparation of wall materials. Neutral mono-saccharide composition was determined as described previously (Zhanget al., 2012). The cellulose content was determined as described previously(Updegraff, 1969). For the extraction of acetyl-xylan, the destarched wallresidues was depectinated using 1% ammonium oxalate and then delignifiedwith 11% peracetic acid at 85°C. Acetyl-xylan was extracted twice withDMSO at 70°C and precipitated with the ethanol:methanol:water (7:2:1) for3 d at 4°C. The pellets were rinsed with anhydrous ethanol four times andcollected by centrifugation. The acetate content was analyzed using theMegazyme Acetic Acid Assay Kit (K-ACET, Megazyme) as described pre-viously (Gille et al., 2011b). The experiments were carried out with at leastthree biological replicates.

Western Blotting

Young internodes from mutants and wild-type plants were ground inliquid nitrogen andhomogenized in extraction buffer (25mMTris-HCl, pH7.5,0.25 M Suc, 2 mM EDTA, 2 mM DTT, 15 mM b-mercaptoethanol, 10% glycerol,proteinase inhibitor cocktail [Roche]). After centrifugation at 10,000 g for20 min at 4°C, the supernatant was centrifuged at 100,000 g for 2 h at 4°C toobtain a microsomal pellet. Twenty micrograms of microsomal protein wasseparated by SDS-PAGE gel electrophoresis and transferred to a nitrocellu-lose filter membrane. The proteins were probed with primary antibodies(1:1,000) and the secondary HRP conjugated antibody (1:5,000; Sigma). Thesignals were developed with the ECL plus western Blotting Detection Systemkit (GE Healthcare).

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For Suc density gradient centrifugation, wild-type seedlings were homog-enized in extraction buffer (250 mM sorbitol, 50 mM Tris-acetate, pH 7.5, 1 mM

ethylene glycol-bis-(2-aminoethylether)-N, N, N’, N’-tetraacetic acid, 2 mM

DTT, 1 3 protease inhibitor [Roche], 2% [w/v] polyvinylpyrrolidone, and4 mM EDTA). The samples were then centrifuged at 10,000 g to remove tissuedebris. The supernatant was centrifuged at 100,000 g for 2 h. The pellets weresuspended in a microsome suspension buffer (5% [w/v] Suc, 20 mM Tris-acetate,pH 7.5, 0.5 mM EGTA, 13 protease inhibitor and 4 mM EDTA) and subjected tocentrifugation in 10% to 50%Suc linear gradient, 20mMTris-HCl (pH 7.5), 0.5mM

EGTA, 13 protease inhibitor, 4 mM EDTA at 100,000 g overnight in a SW41Tirotor (Beckman) for fractionation. Fractions were collected and separated bySDS-PAGE and subjected to western blotting as described above.

OsTBL1 and IRX14 polyclonal antibodieswere produced inmice and rabbitsagainst apolypeptide consistingof aminoacid residues 100 to150ofOsTBL1andamino acid residues 456 to 524 of IRX14, respectively. The antibodies werepurified through affinity chromatography before use. Anti-BiP, anti-PIP1s, andanti-His antibodies were purchased from Agrisera and Sigma.

Gene Expression Analyses

To examine the expression ofOsTBL1 andOsTBL2, total RNAwas extractedfrom young internodes using the Plant RNA Purification Reagent (Invitrogen).cDNA was synthesized from RNA using the Reverse Transcription System kit(Promega). The expression level of OsTBL1 and OsTBL2 in wild type, mutants,and knock-down and overexpression plants was examined by qPCR with aCFX96 Real-time System (Bio-Rad) as described previously (Huang et al., 2015)by using the primers presented in Supplemental Table S4. Rice HNR or ZCF61gene was used as an internal control.

Subcellular Localization Analyses

The coding sequence ofOsTBL1was cloned into pCAMBIA1300 between theCaMV 35S and NOS terminator in frame with GFP. The resulting vector wasintroduced into A. tumefaciens EHA105 and transformed into 1-month-oldNicotiana benthamiana leaves together with a vector harboring the cis-Golgimarker Man49-mCherry. The fluorescent signals were observed with a con-focal laser scanning microscope (Leica TCS SP5) after 3 days. Colocalizationanalysis was performed using the line profile of the Zen software package(Zeiss). For immuno-electron microscopy, 3-d-old wild-type rice root tipswere cryofixed by high-pressure freezing using a Leica HPM100 and infil-trated with 2% uranyl acetate in acetone at 290°C for 48 h. The samples wereembedded in lowicryl HM-20 resin using a Leica AFS2. Ultrathin sections(80 nm) were prepared and subjected to immune-labeling with the anti-OsTBL1/2 antibody at a 1:1,000 dilution and 10 nm colloidal gold-conjugatedgoat antirabbit IgG (Abcam) at a 1:20 dilution. After poststaining in 1% uranylacetate (70% ethanol), the images were acquired with a transmission electronmicroscope (Hitachi HT7700) equipped with a CCD camera (Gatan 832).

Recombinant Protein Expression

The coding sequence ofOsTBL1without the transmembranedomain (84–502amino acids) were fused with a 63His tag and cloned into the pPICZa vector(Invitrogen) to obtain OsTBL1 recombinant protein. After transformation intoPichia pastoris X-33 cells by electroporation following the manufacturer’s in-structions, the high-yield clones were screened and induced in the culturemedium containing methanol. The recombinant protein was purified using aHisTrap HP column with a gradient of imidazole (0–500 mM) using an AKTAFPLC system (GEHealthcare). The recombinant protein is examined bywesternblotting with the anti-His antibody. Protein concentration was determined bythe Bio-Rad protein assay (Bio-Rad).

Enzymatic Assays

For examinationofOsTBL1acceptor substrate, xylan, pectin, xyloglucan, andlignin were purchased from Sigma and Megazyme. Purified recombinantprotein (3 mg) was incubated with 30 mg of the above-mentioned acceptorsubstrates in sodium phosphate buffer (50 mM, pH 6.8) containing 200 mM

acetyl-CoA (Sigma) and 0.7 mM3H-radiolabeled acetyl-CoA (PerkinElmer) at

25°C for 16 h. After applying 200 mL of 2 M sodium chloride to stop the reaction,the products were precipitated in 500 mL ethanol and filtrated through 0.45-mmnitrocellulose filter (Millipore). The incorporated signals were quantified by a

liquid scintillation spectrometer (1450MicroBetaWallac TaiLux; Perkin-Elmer).To determine the preferred acceptor substrate of OsTBL1,we incubated 0.25mM

xylooligosaccharides, such as Xyl (Sigma), xylobiose, xylotriose, xylotetraose,X5, or X6 from Megazyme, with 10 mg of purified recombinant OsTBL1 in theabove reaction buffer for 40 h. The resulting acetylated productswere examinedby LC-QTOF-MS. To analyze the acetylated X5 by MALDI-TOF MS, 3 mg ofpurified recombinant OsTBL1 protein was incubatedwith 0.25mMX5 and 1mM

acetyl-CoA in sodium phosphate buffer (50 mM, pH 6.8) for 16 h. The reactionproductsweremixedwith amatrix solution (10mg/mL2,5-dihydroxbenzoic acid)and spotted on the target plate with 0.5 mL of 5-chloro-2-mercaptobenothiazole(10 mg/mL). The positive ion spectra were recorded on an ABI 4700 ProteomicsAnalyzer (Applied Biosystems) using a 200-Hz frequency of Nd-YAG laser op-erating at a wavelength of 355 nm. Averages of 2,500 shots were used to obtain theMS spectra in a reflectronmode. Cellulohexaosewas included as internal standardfor quantification. To examine the OsTBL1 activity in a time-dependent manner,the reactions were performed as MALDI-TOF MS analysis except incubation fordifferent time (0 to 80 h). The ratio of acetylated X5 to total X5 added in each re-action was measured. To determine the kinetics of OsTBL1 the purified protein(3 mg) was incubated with X5 in the presence of 1 mM acetyl-CoA for 16 h. Thereactionwas stopped and applied to aMALDI-TOFmass spectrometer to quantifythe acetyl residues on X5. The steady-state parameters Km and Vmax were deter-mined by Michaelis-Menten plots using nonlinear curve-fitting in Origin 9.1(OriginLab). ForMALDI-TOF/TOF tandemmass spectrometry analysis, collision-induced dissociation mass spectra were obtained on an ABI 4800 ProteomicsAnalyzer (Applied Biosystems). The parent ion was collided in the collision-induced dissociation cell under high collision energy (1,000 V). The experimentswere carried out with three biological replicates.

NMR Analysis

NMR characterization of cell wall residues from the wild type and mutantswas performed as described previously (Cheng et al., 2013). Twenty mg of cellwall residue was milled with a planetary ball mill PM 200 (Retsch) and dis-solved in 0.6 mL of DMSO-d6 (D, 99.9%; Sigma) supplemented with 1-Ethyl-3-methylimidazolium acetate, a kind gift of Prof. Suojiang Zhang, Instituteof Process Engineering, CAS. The samples were subjected to NMR analyses.1H-NMR and HSQC NMR spectra were acquired at 298 K on an Agilent DD2600MHz NMR spectrometer equipped with a gradient 5-mm HCN triple res-onance cold probe. The Agilent standard pulse sequence gHSQCAD was usedto determine the one-bond 13C-1H correlation in wall residues. All the 1H-13CHSQC spectra were collected using a spectrum width of 10 ppm in the F2 (1H)dimension and 200 ppm in F1 (13C) dimension. 20483512 (F23F1) complexdata points were collectedwith the receiver gain set to 30, and 64 scans per FIDwere accumulated with an interscan delay (d1) of 1 s. The spectra were cali-brated by the DMSO solvent peak (dC 39.5 ppm and dH 2.49 ppm). The NMRdata processing and analysis were conducted using the MestReNova 10.0.2software.

To examine the regiospecificity of OsTBL1, 0.5 mg X5 and 600 mg OsTBL1recombinant protein was incubated in potassium bicarbonate buffer (50 mM,pH 6.8) containing acetyl-CoA (1 mM). After 40 h of incubation at 25°C, thereactions were stopped and freeze-dried. The products were dissolved inD2O and loaded onto an Agilent DD2 600MHz NMR spectrometer. TheAgilent standard pulse sequence gCOSY was used to determine the 1H-1Hcorrelation.

Inoculation of Leaf Blight

Todetermine the resistance to leaf blight disease, three tofive fully expandedleaves of 3-month-old wild-type, ostbl1, ostbl2, and the tbl1 tbl2 plants wereinoculated with water or Philippine Xanthomonas oryzae pv. oryzae (Xoo) racesPxo61 and Pxo145 according to the leaf-clipping method (Kauffman et al., 1973).The lesion length was measured 2 weeks after inoculation (Zhong et al., 2003).

Saccharification Assay

One milligram destarched wall material was suspended in water andboiled for 1 h to inactive endogenous enzymes. After cooling down, the di-gestion was performed with an enzyme mixture containing 2 mL Ctec2 en-zyme (Novozymes) in 50 mM citrate buffer (pH 6.2) at 50°C with shaking at220 rpm for 3 and 24 h. The released sugars in the supernatant were mea-sured by reading the A540 on an ELISA reader (Tecan) as described previ-ously (Huang et al., 2015).

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Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers AK060755 (OsTBL1) and AK065582(OsTBL2).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. OsTBL1 and OsTBL2 are highly conserved.

Supplemental Figure S2. The gene expression analysis and phenotypiccharacterization of the transgenic plants.

Supplemental Figure S3. Anatomic analysis of internodes.

Supplemental Figure S4. Purification of OsTBL1 recombinant protein.

Supplemental Figure S5. Coexpression analysis of OsTBL1.

Supplemental Figure S6. Examination of the expression of OsTBL1 andOsTBL3.

Supplemental Figure S7. Saccharification analysis of the wall residuesfrom wild type, ostbl1, ostbl2, and tbl1 tbl2 internodes.

Supplemental Table S1. TBL gene family in rice.

Supplemental Table S2. Expression pattern of TBL genes in rice.

Supplemental Table S3. Monosaccharide composition and cellulose con-tent of cell wall residues from the wild type, mutants and transgenicinternodes.

Supplemental Table S4. Primers used in this study.

ACKNOWLEDGMENTS

WethankZhang S. (Institute of Process Engineering,CAS) for providinguswith1-Ethyl-3-methylimidazolium acetate; Cao X. (Institute of Genetics and Develop-mental Biology, CAS) for providing the artificial miRNA vector; ZhaiW., Zhao J.,and Liu P. (Institute of Genetics and Developmental Biology, CAS) for providingthe Philippine Xoo races and the help on inoculation; and Sun S. (Institute ofMicrobiology, CAS) for the assistance in MALDI-TOF experimentation.

Received October 20, 2016; accepted November 16, 2016; published November18, 2016.

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