2020 - Proteomic Screening For The Identification of Proteins Involved in Resistance To

Physiological and Molecular Plant Pathology 113 (2021) 101562 SS aa Physiological and Molecular Plant Pathology Journal homepage: wr. elsevier-comilocatelompp \« Proteomic screening for the identification of proteins involved in resistance to Xanthomonas campestris py. malvacearum in cotton Ivonaldo Reis Santos", Thuanny Borba Rios“*°, Mariana Rocha Maximiano””*, Witton Macedo Coutinho “, Liziane Maria De Lima", Luciano Paulino silva", Osmundo Brilhante Oliveira-Neto , Angela Mehta {Entrape Renee Gedo «Bien PBL A. W/5 None Fal CEP 70770917, Bea, DF, Bre Pepa de Ps Crasngas om Cane Beliear (Blogs Nala), Utena de Bria Ft de Cor Bngeas Campus Univers Day "Png de Bi ran en Cle Bigs ase IP/ Gee Besa Ueda tra de i eo Camp esis, Ra mre Rene So Pe, ede era CEP 9606 800, MO, Bra Entra aod Ondo Goa, 1143 Comma, Conga Grade, CEP 58426055, PB, Bras * Doporamont de Bogie «Blog Noll Ful de Media, FAIPLAG SICA AR2, Bu, DF CEP 72460 000, ad ARTICLE INFO apsTRact ower | this sty, we Henfed protein in orton asocintd with resisance ro Bateril Bligh (BR) by sing two. ctrl Dg imensional elecuophotesi (2 DE). To seach for proteins that may tigger resstance/defense response, ete 20 we analy Gasypunn hsuwun plants at 24 and 120 h fter inoculation With Xonthomonas canpess ‘nataceain (Xe). Photosrthese elated proteins sowed pronownced vedicton in te Kem inoculate plats ‘These results indicate har the resistance respons in cotta ineoives the eduction of poten abundance to cope with ines caused by Xen and suppost cur previous studies showing dat che negative regulation of photo sythess i eruelal for relstance to allow an active defense vesponse 1. Introduction ‘The Xanthomonas genus comprises many plant pathogenic baceri, Which affect a great variety of crops of social and economic relevance. Xanthomonas campesrs ps. malvacearum (Xem) is the enusing agent of ‘cotton bacterial blight (BB) considered one of the most important dis ‘enses, causing severe production losses [1,2]. The symptoms of cotton BB range from angular spots to burn of planets. The mst conunon ‘control measures of this disease include the use of healt seeds, bio logical contro, erop rotation, an especially resistant plants, considered the most efilent method A total of 18 of the main genes or polygene complexes of BB ress: tance in coton (B genes) have been investigated. Among these, the B12 ‘gone, which confers resistance Xeni race 20, and the Bom gone ean be highlighted [5]. The pyramiding of B genes has been successfully used to produce lines with high resistance against multiple races of Xem. For example, pyramiding B2 with BS and other polygenic complexes [1,9] provided substantial protection against all races of Xem identified in the Us [o]. The $295 genotype resistance of Gosypimhirsurum used in that study is genetially linked to the locus BS and may iavelve a single dominant gene closely associated with this locus or, more probably, @ mplex luherted as single locus [71 One of the aproaehes to identify Key molecules involved in plant psthogen interctions is proteomies. Proteomic analysis represents & Valuable approach forthe sty of differential protein abundance ait Ing atthe identification of proteins related to plant defense processes against various types of pathogens. This method has been highly wset for the understanding of physiologieal processes and identification of Atevaions Xem, Xauhonanascanpesrs py. malbocearun; 8, Ctton bscteral blight -DE,rwo-émensionaleleophotesis; NYG, Nuslent Yeast Glycerol hal, ours afer Inoculation; Gls, Gene Menier: NCBL, National Center for Biotechnology Information GO, gee ontology; ROS, reactive oxygen species PME, Depide mace Bagerpvnting, TFA, luorocere acd; MALDL, enatix assed laser desorption oniaton; TO, saeof Fgh; MS/MS, tandem mass spectionery. * conespouang tho Email adresses: ivonaldorissanos@ynailcom (LR. Santos) thuannyborbaGgmallcom (TB, Rios), mariananainianor@gmaileon (NLR, Maxinian), ‘wutencoutshogtembrapa br (W.M. Coutinho), liane ina@embiapa.br (LM. De Lina), lciano pauliaodtenbrepaby (LP. slva) osmunde bilhantegg.cnpa by (OB Olveir Neco}, angela mehia@embvapa.s, angela nehiatembeapa b (A, Mehta). ps /do.or/10.1016/)pmpp.2020.101562 Received 12 Apil 2020; Received in revised fons 30 July 2020; Ascepad 5 Oster 2020 Availabe online 9 October 2020 ‘0845'5765/0 2020 Eee Lid, Al rights reserved,proteins related to pathogen resistance [12 In this contest, the objective ofthis study was to identity proteins in cotton ivolved i the resistance to Xen, sing the classical 2.DE pro: teomie approach associated with mss spectrometry. The results eb tained inthis study contribute ta beer understanding ofthe processes involved in response to Xem and can help breeding progranis in the developnent of resistant cultivars, 2. Materials and methods 2.1. Plant material and inoculations Gossypium hinunn 5295 genotype, resistant 10 Xem race 18, was ‘obtained from Embrapa Algodio, Campina Grande (PB), Brazil, and ‘ultivated in greenhouse conditions at Embrapa Recusos Geneicos © Biotecnologia, Brasilia DF, Brazil. Nem was grown in NYG (Nutrient Yeast Glycerol) medium [1] at 28 °C for 24 h, suspended in stele sine solution (NaCI 0.8535) and the optical density was adjusted (0 ‘Aso = 0.6 for inoculation using a syringe. At 45 days after sowing, nine plants were inoclated with Xeni and nine plants vith sterile saline so lution (mock inoculation), which were considered as the control eon- dition. A total of three biological replicates (samples) per condition Cnoculated and control were used, orallzing 18 plants. The sampling of| the plant material for the analyses was carried out at 24 and 120 hours ‘after inoculation (ha). All collected leaves were ground in liquid mi trogen and stored at ~80 °C until futher use fr protein extraction 2.2, Total protein extraction and two-dimensional electrophoresis (2-DE) ‘oral proteins were extracted ftom 0.1 g of tissue using the phenol method according to Carmo et al. [12]. Proteins were suspended in solubilization buffer (7 Mure; 2M dhiouren; 4% (n1/¥) CHAPS: 29 IPG Duifer with pH 4-7; and 40 mM DTT). The quantification of procein ial ‘concentration was performed using Bradford reagent (BioRad) following the manufacturer's instwetions, Hydration of IPG strips Cnumobiine DrySerip, 13cm, pH 3-10 NL) was performed with approximately 600 ug ‘of protein for a period of 16 h. Protein separation by soelecee point ‘was performed ssing IPGphor Ill Electrophoresis system (GE Heath are) for about 5 h and 30 min, with changes in voltage and amperage carried out according to the manufacture's manual. After the electro: phoresis, dhe IPG strips were equilibrated for 20 ai in equilibration buffer 1.5 M pH 8.8 Tris HCl; 6 MI urea; 30% glycerol; 296 SDS, and 1% bromophenol blue) containing 1 MC DTT, followed by 20 min in equi bration buffer containing 2.5% iodoacetamide, ‘The second dimension was performed in 12% polyacrylamide gel ‘and electrophoresis was cared out in vertial system using glycine buffer 20 mit Tes HCL; 0.102 mt glyeine; and 0.1% pH 83 SDS) and molecular weight marker “Benchmark Protein Ladder", according to Carmo et al [12]. Electrophoresis was performed for a period of approximately 6 hand 30 min at room temperature and at least three ‘ges (RI, R2, and RS) per condition (Inoculated and contol) of each ‘sample (24 and 120 hai) were prepared. Proteins were stained with Colloidal Coomassie Blue G-250 (0.1% w/v Coomassie Blue G-250; 2% '¥/¥ phosphoric acid; 10% w/v ammonium sulfate; and 20% v/v meth ‘anol) and the gel replicates were digitalized with the ImageScanner IL (GE Healthesre). 2.3. Image analysts, proen digestion, an protein entfeation by mass ‘spectrometry Using the 2:DE approach, the G. hirsunun-Xem interaction was ‘analyzed at two time points (24 and 120 ha). Three gels (one from each biological replicate) with the highest spot resolution and homogeneity wore selected and used for data analysis in the Image Master 2D Plat ium? software (GE Healthcare). Spots were automatically detected by the software, ind when necessary edited mannally. Dace were Piya and Mole Pla Pag 1192021 101562 normalized by expressing protein abundance as relative spot volume. Automated matching was performed and manual spot detection was performed when necessary. Two statistical tests were used to validate Aiferentiat spots. First, the r? test was used to evaluate the correlation berween the gels and subsequently the ANOVA test (p < 0.05) ta determine the differentially abundant proteins. ‘The differential protein spots were excised from the gel for digestion, ‘The gel fragments were dried, rehydrated with 1 yg of trypsin (Sigma Aldrich) and incubated at 37 °C overnight. A total of 1 iL of each digestion was mixed to 1 pL of alpha-cyano-4-hydroxyeinnamic acid matrix (10 ig min 30% of acetonitrile ané 0.1% TEA) and applied to ‘a Anchor chip target plate Bruker Daltonies) according 1 Carmo etl 021 Hydrolyzed peptides were analyzed by MALDITOF-TOF UltraFlex sass spectrometer (Bruker Deltonies) operating in positive reflector (Ms) and LIFT (MS/MS) niodes. The list containing mass profiles was generated by the software FlexAnalyss 3.8 software (Bruker Daltonics). All searched MS and MS/MS peak lists were conducted using the MASCOT platform (hip//2ww.snarixscience.com) with the NCBINE protein database and the Viridiplantae taxonomy option. The parame: ters used in MS were: mass tolerance in 150 ppm and one missed cleavage; carbamidomethylation of cysteines considered as fixed ‘modifieaton; oxidation of methionine as variable modification; and the cutof® value for the probability based Mowse score calculated by MASCOT (p < 0.05) was used to accept identifications. For MS/MS data, the following parameters were used: peptide mass tolerance at 150 ppm: [MS/MS fon mass tolerance at 0.6 Da; allowance of one missed cleavage; fd change state 1 24, Gene ontology analysis Afier protein identification, the GIs (Gene Identifiers) were used to obtain the FASTA sequences in the NCBI database and a zene ontology (Go) analysis was performed aiming to understand the biological pro cesses in which these proteins are involved using Blast2GO% software (141 This analysis also helped to determine possible localization, mo. lecular functions, and allowed the generation of KEGG maps, showing the metabolic pathways associated. The differentially sbundant proteins Were clustered into eight categories according thelr GO terms and te cording tothe literature. 3. Rosulls and discussion 1, Hypersensitivity reaction In cotton caused by Xem In order co verify the symptoms in G. hrsunun $295, plants were Ingeulated with Xem race 18 nd compared (othe susceptible reaetion of genotype Acala 44, At 48h after infection a clear hypersensitive reaction ould be observed in S295, while in the suscepdble genotype, soaked lesions were present at § days after inoculation (Vig. 1), as previously Aetennined by de Sousa et al. [15], 8.2, ZDE naps and identification of cfferentaltyabundane proteis In this su, proteins of G. lrsuzum $295 resistant genotype were evaluated by 2-DE at two time points (24 hai and 120 hal). We consi. red 24 hal as the onset of infection and 120 hai asa later stage of| Infection, The present analyses revealed approximately 500 spots per sel, with molecular mas ranging from 10 to 160 kDa and pf from 3 to 10. When the protein profile ofthe Xent inoculated plants, collected at 24 bi was compared to the control condition, 93 protein spots showed statistically significant varintion, including 47 decreased and 26 Increased proteins, 17 unique spots to the control, and 3 unique to Xen inoculated plants, A total of 34 diferental protein spots were success fully identified by MS (Supplementary Table 1), including 28 decreased rnd 6 increased in Xem-inoculated plants (is. 2nd Fig. 9.RSet Phyo! nd Mole ln Paley 1192021) 01562 Fig. 1 Cotton leaves of Ala 44 a5 days afte Inoelacon (A) an 5295 a2 days inoetaton (8) esplaying sscesbiity and ypersensive een CHR), espectvely after inoculation with Xowhomonas cones py. mataceana ace 18. 298 Con 24 hat 285 cto 24 eee 1p ct 10 = r= Fig. 2. 2DE analysis of proteins obsaine fiom plants inoculate with Xem at 24 alter inoculation (hal an lathe contol condition. The numbers show the lifeteial proteins identifi the symbols + and ndeate ineiased and decreased proteins respectively. Sprs witout signals indicae unique proteks, Fig. 5. Difeensal abundance ofthe Henrifedpoteins. The values ate based onthe spor latest ato ln plants inoculated with Xcm a 24 after inoculation al) ‘compared tothe conto contin; ted bas inate decreased poten and green bars indicat inereasedproeins. (For inerpreacon ofthe reerenes to colour in this gate legend, the vader i eee othe Web version ofthis ale)In the analysis performed nt 120 as, 52 protein spots showed sta Ustically significant variation, including 20 decreased proteins, 18 Increased, 13 unique to the control, and 1 unique to Xen inoculated plants. A total of 26 differential protein spots were identified (Supple ‘nentary Table 2), including 16 decreased, 8 increased, and 2 unique t0 the control (Vis. and Fig.) Interestingly, the result showed that most ‘of the identified differeatially abundant proteins were decreased in the ‘ent inocolated plants wien compared to control plants at 24 and 120, hi. These results suggest thatthe resistance response in cotton involves the reduetion of protein abundance to cope with infetion caused by ent, Most ofthe deetensed proteins identified at 24 and 120 hal, were associated (0 photosynthesis, indicating that this modulation may be Important to influence the eontainnient of Xem. This observation Is ‘consistent with previous reports tha sggest that a negative regulation ‘of photosynthesis is erucal for a resistance response [0], which may be associated co te ned forthe activation of defense mechanisms [16,17] 3.3. Functional annoration by gene ontology ‘To understand the biological processes in which these differentially ‘abundant proteins identified are involved, a gene ontology (GO) search ‘was performed from the results obtained inthe 2-DE analysis at 24 and 120 hal. The biological processes were grouped according to GO and the literature (Supplementary Table 8) into 8 categories, which inelde proteins involved in detoxification, localization, biological regulation, Imetabolle process, single-organism process, response to stimulls, cellular process, and regulation of biological process (Fig. 6). Ie was interesting to observe that at 24 la, all biological processes in which these differentially abundant protelus identified are involved were decreased, while all biological processes were inereased at 120 hat Some proteins, within these categories, considered as having a crucial ‘ole in the bacterial containment are highlighted and their biological, toes are discussed further. 14, Decrease of photosynthesis-relted proteins for defense activation Comparative analysis of protein profiles of 6, hisurtum inoculated ‘with Nem and the control condition evaluated in the present study showed thar he photosynthedie achinery was severely affected. Tt i [knowin thar photosynthesis is strongly modulated during plant-pathogen, interaction [J] and that reduction of this process can deprive the |$206 Control 120 ha 0a 3 ——______#._4 Phyo! nd Mole ln Paley 1192021) 01562 pathogen of nutrients, which isa benefit forthe host [19] Inthis study, twas possible to observe chat most ofthe proteins were negatively regulated, most of which were related to pliotosyathesis Including ribulose biphiosphate carboxylase activase (spots 199 and 98, Supplementary Table 1; spot 204, Supplementary Table 2), ribulose biphosphate carboxylase large chain (spot 145,. Supplementary “Inble 1), ribulose biphosphate carboxylase (spot 97, Supplementary ‘Table 1; spot 156, Supplementary Table 2), ATP synthase beta subunit (spot 105, Supplementary Table 2), mbisco large subunit-binding pro: tein subinit pot 99, Supplementary Table 1), glyceraldehyde Phosphate dehydrogenase B, eloroplastic (spot 267, supplementary “Inble 1), and photosystem II polypeptide, oxygen involving enlancer 1 {spot 185, Supplementary Table 1). These rests show that the photo: synthetie appara related to defense responses ro the paren. Studies have shoven that the negative regulation of photosynthesis related proteins is associated to che deprivation of photosynthesis and other processes, suc as increased respiratory metabolism activities and eatboliydeateearriers, to inilate processes necessary for plant defense {16,181 by che induction of plant defense gene expression [20]. Stdies conducted by Santos et al. [21] shoveed that infection by Xanthomonas campestris p. campestris in the moderately resiscant cultivar Astus Plus of Brusca oleracea resulted in decrease in the abundance of proteins related to photosynthesis, Indicating # strong Tink 10 te defense response. Similarly, Shuai etal. (22) also demonstrated in the sugar ‘anie-Pusarium vericlioides interaction that the photosynthetic rate, stomatal conductance and transpiration rate of che sugareane plants were significantly reduced, showing a strong link with plant defense process. It has also been shown that resistant ree plants infected with Xanthomonas oryzne pv. oryzae had their photosynthetic capabilities compromised, evidencing significant reductions in chlorophyll content and downregulated expression of genes related to photosynthesis [2°]. These results indicate that the resistant interaction of G, hiurtum em sppears to utlize similar defense mechsnisms to inhibit pathogen growth, Ths, che resus obtained i this study suggest that the decrease im the photosynthetic activity of Xen-inoculated resistant plants of 6, hisurtum is primarily a defense mechanism in the attempt to contain the pathogen. In studies performed by Ribeiro et al. (9, similar results were also obtained in rusia oleracea Unio cultivar resistant 0 Xan ‘homonas canpestis pv. campestris within 24 hai, showing that the negative regulation of photosynthesis is related t0 an active defense response {5205 Inoculated 120 hai Fig. 4. 2DE analysis of proteins obcained Rom plans inceuated with Xem ar 120 after Inoeatlon (ha) and inthe conto condition. The numbers show the slifereial proteins identifi he symbols + and Indicate increased and decreased proven, respectively. Spo without sigal india un ve pres.RSet Piya and Mole Pla Pag 1192021 101562 Fig. 5. Ditfrentil abundance ofthe identified proteins. The vas ake based on the spot intensity ratio in plants inoculated with News at 120 ater inocation (hai) compare eo the contol condition; red bars incae decreased proteins and green bars indiae increase proteins. For imerpresaton ofthe seerenes 0 ‘ol in this Fgue legend, he ede referted co che Web version of his ariel) a ca » how Fig. 6. Functional categorization of the i biologie process of proteins Menied Dy — — -MALDITOF-TOF in $205 genotype A Plans a te ‘ove Inoculated wit Xem at 24 hae noel — i —— =o thn tt compe thet naan ! : — 'B Plants inoculated with Xem 120 al — — 5 ee mms ona 0 the consol condone bas Ile fase bilge pees eed oe i ae : eves (For nerpieton ofthe eferences DC ——« aa tein tie fgre eede vear Irene oe Web eso of ise) Pe = 25, Defense lated pres bored metabo oxidative ses (261. Our fesuts show tht the presence ofthese pro teins indicates strong connection to the process of defense response. Proteins modulated during the Xem-cotton interaction showed that Thetefoe, i¢ is possible thatthe activation of antioxidant proteins is besides the photosynthetic apparatus, metabolism undergoes several related 0 the coutrol of oxidative stress levels triggered during Xen. ‘changes in plants infected by Xanthomonas, Proveins iavolved in meta. infection, olism, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (por 243, supplementary Table 2, spot 267, Supplementary Table 1) 4, Conetusion ‘and alcohol dehydrogenase (spot 115, Supplementary Table 2) were decreased. Stdies have shown that proteins such as GAPDH are Proteomic analysis is tool that facilitates the study of global protein increased in susceptible interactions evidencing thactheiraeeumulation expression and provides a large amount of information about the in ‘can lend to defense response [2]. Intriguingly, the GAPDH identified in vidal proteins involved in specific biological responses, The proteins this study showed a more pronounced decrease at 24 hal when qyalnated in this study have important roles related mainly to photo ‘compared to 120 hai. Possibly, atthe beginning ofthe infection a higher synthesis and defense. The results showed that most proteus identified Feditetion in metabolism is necessary 10 aetivate defense responses 10 ¢ 24 and 120 hai were associated to photosynthesis, We suggest thatthe contain the pathogen. As the defense response progresses, the plant decrease in photosynthetic activity is of great importance forthe defense begins to reestablish ts cellular processes (191 response, The results obtained in thie study have an importa impact in advancing the eurrent knowledge regarding protein expression during 36, Protein accumulation assclaed with detoxication the interaction between cotton and Xem and particularly in under stauding the biological processes involved during dhe defense response. In the present study, two proteins were related to detoxification This information is relevant since i ean support the improvement and Inchuding 2.Cys peroxiredoxin chloroplastc (KIB62039,1) and Perox-__ Seneretion of new cultivars of eoton more tolerant or resistant to Xem. lredoxin chloroplast (Q58186.1) (Supplementary Table 3). Peroxr- ‘edoxins ae important for the contol of peroxide ané thiol levels i the Author conteibuti cells and respond to redox stress to regulate homeostasis [25]. The ac tivity of Prx type 2 Cys is relate to cell protection against reactive ox Ionaldo Santos performed, analyzed, and interpreted data and ‘ygen species (ROS), protecting the photosyntherie apparatus ftom wrote the mianuseript. Thuanny Rios performed, designed experiments‘and wrote the manuscript, Mariana Maximiano assisted in proteomic data analysis. Lziane Maria de Lima and Witton Coutinho designed ‘experiments, Luciano Silva asisted with mass spectrometry analysis. ‘Osmundo Oliveira Neto assisted in plant cultivation and dats analysis. ‘Angela Mehta designed experiments, analyzed data, wrote the nant script, and leaded the work. All the authors tead, revised, and agreed ‘with the content ofthe manuscript. Declaration of competing interest ‘The authors declare that they have no known competing financial {Interests or personal relationships that could have appeared to influence the work reported in this pape Acknowledgements ‘This work was sponsored by Embrapa, Coordenacao de Aperfeicos ‘mento de Pessoal de Nivel Superior. CAPES, Consetio Nacional de Desenvolvimento Cientiico e Teenoldgico -CNPq, and Fundagao de "Amparo Pesqisa do Distrito Federal FAPDF. Appendix A. Supplementary data Supplementary dao this article can be fond online at ips ‘0rg/10.1016/5.pmpp.2020.101562. References (1 At volouoks,p mamey, Dano A Jallou Mvane, th Moc ‘Soning at ehareteraton af Coop tm upton nae gene {ene ote antaningsanpapmcenon meco,Ps (21 Adalat, 4 Champion, M Noe, acter Big of oton, Pijtopthok Meder. 542018) $20 (51 E-Delaocy 30m Pe Mame, Allo, J Dail tl, Rastance of ction ‘erards Ktcotoons cape pv taracen, AR, Phtopatbol42 (200s) 63-62 (4) LBteterot La, Veralen, WAL han, M. Exenbeg, PE Alcan, Deslopmeat finn bac Bo, le Di 68 (1984) 169 (51 Me Boeabers Ma Boje BLL Phe LM. Vee, Pyranog B genes ‘ito aces Doser Bt nays hie tessa o tet DUD, Pyeoptloy 10 (2014) 098-1097 (61 tneerg ML Boles A Saad, A. Hall LBs, Pour nee ogee of cotan wid dierent gone fr base Hight rsa, Paypaigy 92 (2002) 1525-1328 RAs Siva PA. Baron LV Hofmann M Giband, WM. Coutinho, ASS ‘mate licked the BI? poe tht anes retanet race 18 of Xoo ‘ted svacarmincton eso eocned wheter nce gh sie i ms erie Pn Pf 0 2 a Piya and Mole Pla Pag 1192021 101562 181 D6. Ric, ER Can, fa Sai LP Sa 0. eines Net ‘bene learn eine elated potiiind weary geo ack {oc du, Pye Ps aoe Gos) 9-1, tan) Si owe Ho, SSR Anv, O:Abdale, eo, lon sd Cimactenaton of rely expel wea oh pec oitpnCnca gen nc) i repaue wo arctan of eon Mia Mowcea 6 Gon 118. (11) GR Wilt LS. Como, Lt, Sain Ode Obit No, \eeateaton af ras smc raleant baer ce ope to Xanthos cnc pe, camper ect, 3.rowoas 13 10) (18) Laz. chime RO, Red LP. Sv 56. Reo A Mein enon bt tse modal by th sence as AFT mt ra ae i [CWius tenth, Poses 14) 06-00 (03) Deets Bab, F Tue Severe R Bye a Cog of ees Stn in pugs ahomons coments in te iad to ange smi APR EBD 3 (0s) 353 (ay a Bn ea ‘ch attra G05) re doe. {251 Wacom gy Sd on 1D, Seren, WM Ca Pac Short rants sep: masearan smo Br ope Faw htt ote 128 a (06) J.Shae Shin, Wet, Pts an uty rae etl a ince dings icmp acton mith Proporta Fa cl Earn: 2 om) = {271 IFoo Sct Sh Soon Wei RNA ies fears Fn pt 11 Qos) a8 ED. (aa) serge, A. She Ronih, ln poe meet bool: lar Dry mews plat ahoge erations Ep ot 38 (207) 109] Bs Gaovept, Thoma, Gat Zire, 6, Gar, a. Shedng Shen evi poe cston oe, tan) 0 ecnlarpl A line, Te nent ah event ‘sd pisces on oxide sanling rporses Bano aoe ‘Seon, Pans a non: 8 (005) S006 1055 (20 capa tod non val ofs aro eco cdc ve ‘Sinan Sano canes, one Phot S10 G18) 8 tan) {Sina us, 2 Dun, Song. Wang eh Poxyaei elated poten ‘corenbn in sgneae Gring ngsrcane ran vetelider terse SSNS, nso, Pn Gow Rope oa 1. (2xy Kirsten hen, he, Wk ea, tna

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