Functional Genomic in the Search for Disease Resistance in Citrus

Helaine Carrer
Dept. Biological Sciences/ESALQ/USP

Economical Importance of Citrus in Brazil

Employ 400.000 people

> 15.000 farmers > US $1.2 billion 200 million of plants 930 mil ha (81% SP) 22 % of SP agriculture production 504 protected nurseries

Results from narrow genetic basis

Challenges to Citrus Genetic Breeding Botany Juvenility - patterns to differentiate fruit - patterns of growing - limited production Apomixy - genetic e phenotipic fixation - clonal propagation - difficulty to hybridization Sterility - impediment to recombination

Challenges to Citrus Genetic Breeding

Genetics
Incompatibility pre and pos zygotic - obstacle to recombination Heterozygose - hybrid character fixed by apoximy - high F1segregation Long cycle of selection - trees, perennial - Important epigenetic factors - environmental factors X phenotypes

Challenges to Citrus Genetic Breeding

Agronomy
Fruit Quality - complex characteres - environmental effect (herdability) Propagation - vegetative Resistance to biotic and abitotic factors - complex inheritance

Selection of new varieties Disease Resistance

CVC Citrus Variegated Chlorosis

Caused by Xylella fastidiosa Damage: US$ 70 million (cuts, renovation and insect control) Sweet orange: highly susceptible Tangerines and hybrids: tolerant or resistance

Selection of new varieties Disease Resistance

Leprosis

Caused by Citrus leprosis virus (CiLV) Damage: around US$ 100 million to control the vector (Brevipalpus phoenicis) Sweet orange: highly susceptible Tangerines and hybrids: tolerant in the field

Selection of new varieties Disease Resistance

Tristeza

Caused by Citrus tristeza virus (CTV) Damage: virus is endemic Sweet orange: susceptible and highy susceptible depending on the rootstock Tangerines and hybrids: tolerant Poncirus trifoliata: resistence

Selection of new varieties Disease Resistance

Black Spot

Caused by Guignardia citricarpa Damage: avoid fruit exportation Sweet orange: suceptible C. latifolia (Tahiti lime): tolerant

Distribuio de Acessos do BAG Citros

2% 4% 0% 1% 11% 37% 6%

4% 1% 6% 3% 22% 3%

Laranja doce e hbridos Tangerinas e hbridos Limes e hbridos Limo Cravo e hbridos Limo Volkameriano Toranjas e hbridos Outros gneros e espcies

Laranja azeda e hbridos Limas cidas, doces e hbridos Cidras e hbridos Limo Rugoso e hbridos Pomelos e hbridos Trifoliatas e hbridos

CitEST Program
Main goal: Integration
Citrus breeding: Evaluating new scion and rootstock hybrids in the field for disease resistance Citrus genetic: Validation and saturation (SSR and SNPs based on sequence data) of genetic linkage maps from hybrid populations Citrus genome: ESTs database of Citrus species under different stress conditions Functional genomics: expression analysis of genes of citrus and their pathogens

CitEST Program Methodology

Species and varieties: C. aurantifolia (Mexican lime), C. aurantium (sour

orange SP), C. latifolia (Tahiti lime), C. limonia (Rangpur lime), C. limettiodes (Persian lime), C. reticulata (Ponkan), C. sinensis (Pra), C. sunki (sunki mandarin) and P. trifoliata (Rubidoux)

Conditions: infected with Xylella fastidiosa, CiLV, CTV, Phytophthora.

parasitica, drought stress, different tissues (juvenile x mature leaves, fruits at different stages, bark, flowers, roots, seeds)

CitEST Program Methodology

Bioinformatics tools:

CitEST Program Results

Global assembly: final numbers of CitEST program
Species Number of Size of Number of Size of Number of valid reads reads (bp) contigs contigs (bp) Singlets 108,611 51,679 32,637 8,439 8,219 8,756 11,045 8,188 5,216 242,790 854 843 833 850 820 864 803 926 831 14,442 8,307 5,202 1,359 1,642 1,672 2,385 1,560 1,011 37,580 1,002 928 861 775 733 734 684 682 574 17,699 10,566 7,671 4,248 2,685 3,211 3,560 2,980 1,641 54,261 Size of singlets (bp) 848 838 830 846 811 853 807 914 843 Number of Unigenes Size of unigenes (bp) 917 878 843 829 781 812 758 834 740 Redundancy (%)

C. sinensis C. reticulata P. trifoliata C. aurantium C. aurantifolia C. latifolia C. limonia C.limettiodes C. sunki Total

32,121 18,873 12,873 5,607 4,327 4,883 5,945 4,540 2,652 91,821

70.42 63.48 60.55 33.55 47.35 44.23 46.17 44.55 49.15

Only 5ends were sequenced Valid reads = >400 bp and Phred quality >20 Redundancy = [1 - (# Unigenes / # Reads)] # Unigenes = # Contigs + # Singlets Sequencing Efficiency = # Valid Reads / # Submitted Reads Total of sequenced reads = 286,559

CitEST Program Results

Species Citrus sinensis Citrus reticulata Poncirus trifoliata Citrus aurantium Citrus aurantifolia Citrus latifolia Citrus limonia Citrus limettiodes Citrus sunki All species

Global assembly: final numbers of CitEST

#Sequenced reads

Average size of reads (bp)

MB sequenced

Number of Average size unigenes program of unigenes (bp)

MB unigenes

Coverage of the genome n (%) (*) 8.03 4.51 2.96 1.27 0.92 1.08 1.23 1.03 0.54

106601 52679 32637 8439 8219 8756 11045 8188 5216 241780

854.22 842.63 832.73 849.58 819.88 863.55 803.16 926.35 830.92

91.06 44.39 27.18 7.17 6.74 7.56 8.87 7.58 4.33 204.89

32121 18873 12873 5607 4327 4883 5945 4540 2652

917.46 877.63 842.57 828.94 781.11 812.31 757.89 834.3 740.46

29.47 16.56 10.85 4.65 3.38 3.97 4.51 3.79 1.96 79.13

Only 5ends were sequenced Valid reads = >400 bp and Phred quality >20 Redundancy = [1 - (# Unigenes / # Reads)] # Unigenes = # Contigs + # Singlets Sequencing Efficiency = # Valid Reads / # Submitted Reads Total of sequenced reads = 286,559

CitEST Program Results

PFAM of sweet orange
Protein kinase domain RNA recognition motif. (a.k.a. RRM, RBD, or Cytochrome P450 WD domain, G-beta repeat Leucine Rich Repeat Protein tyrosine kinase Zinc finger, C3HC4 type (RING finger) PPR repeat alpha/beta hydrolase fold ABC transporter Myb-like DNA-binding domain Ubiquitin-conjugating enzyme Sugar (and other) transporter Tetratricopeptide repeat DnaJ domain

Total of unigenes in the analysis = 32,121

In silico analysis

HR genes (6300 reads)

metacaspases, Ca++ and K+ channels, NAD(P)H oxidase, peroxidases, superoxide dismutase, nitrate reductase, chorismate mutase, phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, chalcone synthase, isochorismate synthase, lipoxygenase, phospholipase, glutathione transferase, glutathione peroxidase, chitinase, osmotin, thaumatin, permeatin

MAPKs (800 reads)

MAPKs MAP2Ks MAP3Ks MAP4Ks

Transcription factor
Snif1 (535 reads)
Snf1 (sucrose non-fermenting-1) plays a major role in regulating gene expression in eukaryotic cells. This family of regulatory proteins is represented by sucrose nonfermenting-1 (Snf1) protein kinase in Saccharomyces cerevisiae, AMPactivated protein kinases (AMPKs) in mammals and SnRKs (Sucrose non-fermenting-1-related protein kinases) in higher plants

Buchannan et a., 2003

Genes HR: 227 contigs and 174 singletons

Citrus sinensis; CR: Citrus reticulata; C1: leaf cDNA; C3: fruit cDNA; 100: non-infected; 101: infected with Xylella fastidiosa; 102: 30 days after X. fastidiosa infection; 700: stadium 1 of the fruit; 701: stadium 2 of the fruit; 703 stadium 4 of the fruit.

MAP kinases characteristic motifs

(164) C35(123) C40(123) C107(113) C14 (1) C105(120) C60 (1) C47(130) C24(163) C93(141) C50 (95) C37 (60) C89(135) C74(123) C106(140) CR05-C1-103-059-B03-CT.F (34) Consensus(164)

164

170

180

190

200

210

220

230

240

250

267

LTREHHQFFLYQMLRALKYMHTANVYHRDLKPKNILANANCKLKVCDFGLARVAFSDTPMTVFWTDYVATRWYRAPELCGSFFSKYTPAIDIWSIGCIFAEVLT LTPEHYQFFLYQLLRGLKYIHTANVFHRDLKPKNILANADCKLKICDFGLARVAFNDTPTAIFWTDYVATRWYRAPELCGSFFSKYTPAIDIWSIGCIFAELLT -----------------------NVFHRDLKPKNILANADCKLKICDFGLARVSFTDTPSAIFWTDYVATRWYRAPELCGSFFSKDAYLQNC---------------------------PRSSYKANVFHRDLKPKNILANADCKLKICDFGLARVSFTDTPSAIFWTDYVATRWYRAPELCGSFFSKYTPAIDIWSIGCIFAELLT LTPEHHQFFLYQLLRALKYIHSANVFHRDLKPKNILANADCKLKICDFGLARVSFTDTPSAIFWTDYVATRWYRAPELCGSFFSKYTPAIDIWSIGCIFAELLT ---STASISCINFFEDSSISIQQMLFIGDLKPSNLLLNANCDLKICDFGLARPTS----ENEFMTEYVVTRWYRAPELL-LNSSDYTAAINVWSVGCIFMELMN LTNDHCQYFLFQLLRGLKYLHSANILHRDLKPGNLLVNANCDLKICDFGLARTSNG---KNQFMTEYVVTRWYRAPELL-LCCDNYGTSIDVWSVGCIFAELLG LSEEHCQYFLYQILRGLKYIHSANVLHRDLKPSNLLLNANCDLKICDFGLARVTS----ETDFMTEYVVTRWYRAPELL-LNSSDYTAAIDVWSVGCIFMELMD LSEEHCQYFLYQLLRGLKYIHSANVIHRDLKPSNLLLNANCDLKICDFGLARPTS----ENEFMTEYVVTRWYRAPELL-LNSSDYTAAIDVWSVGCIFMELMN FSEAEVRNWCFQVFQGLAYMHQRGYFHRDLKPENLLVSKDT-IKIADFGLAREIN----SRPPFTEYVSTRWYRAPEVL-LQSYLYSSKVDMWAMGAIMAELIT FSEGEIRSFMSQMLQGLAHMHRNGYFHRDLKPENLLVTNDV-LKIADFGLARELS----SMPPYTEYVSTRWYRAPEVL-LQSSSYTPAIDMWAVGAILAELFT LTDDHCQYFLYQLLRGLKYIHSANVLHRDLKPSNLLLNANCDLKICDFGLARTTS----ETDFMTEYVVTRWYRAPELL-LNCSEYTAAIDIWSVGCIFMEIMQ LTREHYQFFLYQLLRALKYIHTADVYHRDLKPKNILANANCKLKICDFGLARVAFNDTPTTIFWTDYVATRWYRAPELCGSFFSKYTPAIDIWSIGCIFAEVLT LTDDHCRYFLYQLLRGLKYVHSANVLHRDLKPSNLLLNANCDLKIGDFGLARTTS----ETDFMTEYVVTRWYRAPELL-LNCSEYTAAIDIWSVGCILGEIMT LTREHHQFFLYQMLRALKYMHTANVYHRDLKPKNILANANCKLKVCDFGLARVAFSDTPMTVFWTDYVATRWYRAPELCGSFFSKVWVAQC------------LT EH QFFLYQLLRGLKYIHSANVFHRDLKP NLLLNANC LKICDFGLARVT FWTEYVATRWYRAPELL L S YT AIDIWSVGCIFAELLT

MAPKs that presented the characteristic motif T(E/D)YVxTRWYRAPE(L/V)

(174) C67(173) C76(150) C86(150) Consensus(174)

174

180

190

200

210

220

230

240

250

260

270

287

EYLAAICEQVLKGLLYLHHEKHIIHRDLKPSKVLIN-HRGEVKITDFGVSAIMASTSGQANTFVGTYNYMSPERIS-----GGKYGYKSDIWSLGLVLLECATGQFPYSPPEQQ PKLAHMASQILKGLSYLHGHK-IIHRDIKPSNLLVNNNNMQVKIADFGVSKIMCRSLDACNSYVGTCAYMSPERFDPDAYGGNYNGYAGDIWSLGLTLLELYLGHFPFLQPGQR PKLAHIASQILKGLSYLHGHK-IIHRDIKPSNLLVNNNSMQVKIADFGVSKIMCRSLDAWNSYVGTCAYMSPERFDPDTYGGNYNGYAGKIWSWGLTLLELYLGHFP------PKLAHIASQILKGLSYLHGHK IIHRDIKPSNLLVNNN MQVKIADFGVSKIMCRSLDA NSYVGTCAYMSPERFDPD YGGNYNGYAGDIWSLGLTLLELYLGHFPF P Q

MAP2Ks that presented the characteristic motif VGTxxYMSPER

(468) C21(454) C10(308) C68(350) C15 (54) C3(112) C109 (22) C64 (81) C91(135) C6 (1) C18 (50) C83 (17) CS00-C1-102-049-B10-CT.F(117) CS00-C1-102-062-D10-CT.F (30) CR05-C3-700-109-C06-CT.F(123) CS00-C1-102-037-B06-CT.F(120) CR05-C3-701-058-D03-CT.F (98) Consensus(468)

468

480

490

500

510

520

530

540

550

560

571

VHRDIKCANILVDANGS-------VKLADFGLAKATK------------LNDVKSCRGTAFWMAPEVIN-----NKNKGYGLPADIWSLGCTVLEMLTSQIPYA VHRDIKGANILVDPNGR-------VKLADFGMAKHIAG-----------HSCPLSFKGSPYWMAPEVIKNSS------GCNLAVDIWSLGCTVLEMATAKRPWS VHRDIKGANILVDPSGR-------VKLADFGMAKHITG-----------QSCPLSIKGSPYWMAPEVIKNSN------GWYLAVDI-----------------VHRDIKGGNILLTDQGE-------VKLGDFGVAAQLTRT----------MSKRNTFIGTPHWMAPEVIPESR-------YDGRV-------------------MHRDIKSENILIDLERKKADGKPVVKLCDFDRAVPLRSFLHTCCIAHRGIPAPDVCVGTPRWMAPEVLRAMHKPN---LYGLEVDIWSYGCLLLELLTLQVPYM IHRDLKSSNLLVDKHWT-------VKVGDFGLSRLKHET----------YLTTKTGKGTPQWMAPEVLRNEP-------SDEKSDVYSFGVILWELATEKIPWD MHRDIKGANILVDNKGC-------IKLADFGASKQAT------------VSGDNSMKGTPYWMAPEVICQT-------GHSYSADIWSVGCTVIEMATGKPPWS IHRDIKGANLLVDASGV-------VKLADFGMAKHLTG-----------LSYELSLKGSPNWMAPEVIKAVMQKDGNPKLALAVDIWSLGCTVIEMLTGKPPWS ------------------------LIYFQFTCCIKLTG-----------LSYELSLKGSPYWMAPEVMQADIQKDGNHRVALLADIWSLGCTVIEMLTGKPPWS IHRDIKGANLLVDASGV-------VKLADFGIGKHLTG-----------LSYELSLKGSPYWMAPEVMQADIQKDGNHRVALLADIWSLGCTVIEMLTGKPPWS VHRDIKCANILVDASGS-------VKLADFGLAKATT------------MNDLKSCKGTPFWMAPEVVN-----SKNDGYGLTADIWSLGCTVLEMLTRRPPYS IHRDLKPENILLFSLDDDVM----LKIADFGLSCTL-----------HPGNYAEKVCGSPIYMAPEVLQFQRYDEKVDMWSVGAILFELLNGYPPFSGRDNVQV VHRDLKSSNLLVDKNWT-------VKVGDFGLSSLKNAT----------YLTAKSGRGTPQWMAPEVLRSEP-------SNEKSDVFSFGVILWELVTASIPWN IHRDLKPENILLSGLDDDVM----LKIADFGLSCSTL----------YPGNYAEKVCGSPLYMAPEVLQFQRYDEK---------------------------VHRDIKGANILVDPHGE-------IKLADFGMAKHMTS-----------CASMLSFKGSPYWMAPEVVMNTN------GYSLTVDIWSLGCTVLEMATFKTTMD IHRDIKGANILTTKEGL-------VKLADFGVATKLTEA----------DVNTHSVVGTPYWMAPEVIEMSG-------VCAASDIWSVGCTVIELLTCVPPYY VHRDIKGANILVD G VKLADFGLAK LT LS SLKGSPYWMAPEVI L ADIWSLGCTVIEMLT K PW

MAP3Ks (MEKK sub-group) that presented the characteristic motif G(T/S)Px(W/Y/F)MAPEV

(139) C29(139) C56(127) C88(135) C59(111) C62 (53) Consensus(139)

139

150

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252

VKNWARQILRGIAYLHGHDPPVIHRDLKCDNIFVNGHLGQVKIGDLGLAAILRGSQHAHSVIGTPEFMAPELYEEDYNELVDIYSFGMCVLEMLTSEYPYSECSNPAQIYKKVT VKLWCRQILRGLLYLHSHDPPVIHRDLKCDNIFVNGNQGEVKIGDLGLAAILRKSHAAHCV-GTPEFMAPEVYEEAYNELVDIYSFGMCILEMVTFEYPYSECTHPAXIYKKVV IKSWARQILQGLVYLHSRDPQVIHRDLKCDNIFVNGHLGQVKIGDLGLAAILRGSKSAHSVIGTPEFMAPELYEEDYNELVDVYSFGMCVLEMFTCEYPYSECANPAQIYKKVT VKGWARQILSGLIYLHSHDPPIIHRDLKCDNIFINGNQGEVKIGDLGLATIMEQAN-AKSVIGTPEFMAPELYDENYNELADIYSFGMCMLEMVTFEYPYSECRNSAQIYKKVS LKKWSRQILEGLSYLHSHDPPVIHRDLKCDNIFVNGNQGEVKIGDLGLAAILAQARSAHSVIGTPEFMAPELYEEEYNELVDIYAFGMCLLELVTFEYPYVECTNAAQIYKKVT VK WARQIL GLIYLHSHDPPVIHRDLKCDNIFVNGNQGEVKIGDLGLAAILR S SAHSVIGTPEFMAPELYEEDYNELVDIYSFGMCVLEMVTFEYPYSECTNPAQIYKKVT

MAP3Ks (ZIK sub-group) that presented the characteristic motif GTPEFMAPE(L/V)Y

(658) C38(629) C28(406) C84(252) C46(302) C16(245) C13(141) C55(141) C65(214) C33 (72) C51(141) C70(147) C49 (47) C39 (96) C79 (80) C99 (95) C23(136) C45 (67) C1(128) C22 (117) C80(145) C12 (181) CR05-C1-100-052-F01-CT.F (1) LT33-C1-003-057-A10-CT.F (36) CS00-C3-702-066-C07-CT.F(130) CS00-C1-100-106-A10-EU.F (3) Consensus(658)

658

670

680

690

700

710

720

730

740

750

761

LHTSHPTIVHRDLKSPNLLVDKN----WVVKVCDFGLSRIKHH---TYLSSKSTAGTPEWMAPEVLRNEPANE--------KCDVYSFGVILWELATLSVPWKG LHQNN--IIHRDLKTANLLMDEN----GVVKVADFGVARVQA----QSGVMTAETGTYRWMAPEVIEHKPYDY--------KADVFSFGIALWELLTGELPYAG VHRLG--LIHRDLKSDNLLIFSD----KSIKIADFGVARIEV----QTEGMTPETGTYRWMAPEMIQHRPYTQ--------KVDVYSFGIVLWELVTGMLPFQN LHQNN--IIHRDLKAANLLMDEN----EVVKVADFGVARVKA----QSGVMTAETGTYRWMAPEVIEHKPYDH--------KADVFSFGIVLWELLTGKLPYEY LHRNN--IIHRDLKAANLLMNEN----GVVKVADFGVARVQA----QYGVMTAETGTYRWMAPEVIEHQPYNH--------RADVFSFGIVLWELVTGKLPYDD LHSHG--IIHRDLKPENLLLTEDL---KTIKLADFGLAREES----LTEMMTAETGTYRWMAPELYSTVTLRQGEKKHYNHKVDSYSFAIVLWELLHNKLPFEG LHANG--IIHRDLKPDNLLLTPDQ---KSLKLADFGLAREET----VTEMMTAETGTYRWMAPELYSTVTLRQGEKKHYNNKVDVYSFGIVLWELLTNRLPFEG VHGLG--FIHRDLKSDNLLISAD----KSIKIADFGVARIEV----QTEGMTPETGTYRWMAPEMIQHRPYTQ--------KVDVYSFGIVLWELITGLLPFQN LHSQG--ILHRDLKSENLLLGED----MCVKVADFGISCLES----QCGSAKGFTGTYRWMAPEMIKEKRHTK--------KVDVYSFGIVLWELLTALTPFDS LHANG--IIHRDLKPDNLLLTPDQ---KSLKLADFGLAREET----VTEMMTAETGTYRWMAPELYSTVTLRQGEKKHYNNKVDVYSFGIVLWELLTNRLPFEG LHSKT--IVHRDVKTENMLLDAN----RTLKIADFGVARVEAQ---NPRDMTGETGTLGYMAPEVLDGKPYNR-----K---CDVYSFGICLWEIYCCDMPYPD LHSKT--IVHRDVKTENMLLDAN----RTLKIADFGVARVEAQ---NPRDMTGETGTLGYMAPEVPTSLFAIP-----Q---RRFSALLLLSC----------IHSQG--VIHRDLKPENVLIDQE----FHLKIADFGIACEEV----YCDALSDDPGTYRWMAPEMIKHKSYGR--------KVDVYSFGLILWEMVAGTIPYEE LHQRN--IIHRDLKTANLLMDTH----NVVKVADFGVARFQN----KGGVMTAETGTYRWMAPEVINHQPYDQ--------KADVFSFAIVLWELVTAKVPYDS LHSKN--IVHFDLKCDNLLVNLKDPSRPICKVGDFGLSKIKR----NTLVSGGVRGTLPWMAPELLSGSSSKVS------EKVDVFSFGIVLWEILTGEEPYAN LHSKN--IVHFDLKCENXLVNLRDPQRPICKVGDFGLSRIKR----NTLVSGGVRGTLPWMAPELLNGSSNRVS------EKVDVFSFGISMWEILTGEEPYAD LHSKN--IVHFDLKCDNLLVNLKDPIRPICKVGDFGLSKIKR----NTLVTGGVRGTLPWMAPELLNGSSSKVS------EKVDVFSFGIVLWEILTGEEPYAN LHRRNPPIVHRDLKSPNLLVDKK----YTVKVCDFGLSRLKAN---TFLSSKSAAGTPEWMAPEVLRDEPSNE--------KSDIYSFGVILWELATLQQPWGN LHEQN--VVHRDIKCANILVDASG----SVKLADFGLAKATT-----MNDVKSCKGTAFWMAPEVVNLKKDGYG------LTADIWSLGCTVLEMLTRRHPYSH LHGKN--IVHFDLKCENLLVNMRDPQRPVCKIGDLGLSKVKQ----QTLVSGGVRGTLPWMAPELLSGKSHMVT------EKIDVYSFGIVMWELLTGDEPYAD LHSIG--LLVLNLKPSNLLLSEHD----QLVLGDFGIPYLLLGRSLSDSDMALRLGTPNYMAPEQWEPEVRGPIS-----FETDTWGFGCSIMEMLTGIQPWFG ----------------NLLVDKN----WVVKVCDFGLSRMKHN---TFLSSRSTAGTAEWMAPEVLRNEPSDE--------KCDVYSFGVILWELCTMQQPWGG LHGKN--IVHFDLKSDNLLVNLRDPHRPICKVGDLGLSKVKC----QTLISGGVRGTLPWMAPELLNGSSSLVS------EKVDVFSFGIVLWELLTGDEPYAD LHTSTPTIVHRDLKSPNLLVDKN----WNVKVSDFGLSRLKHN---TFLSSKSTAGTPEWMAPEVLRNEPSNE--------KCDVYSFGVILWELATLKLPWIG MCVHRMKIVHRDLKSANCLVNKH----WTVKICDFGLSRIITD---SPMRDSSSAGTPEWMAPELIRNEPFTE--------KCDIFSLGVIMWELCTLNRPWEG LH IVHRDLK DNLLV VKVADFGLARI Q MTG GT WMAPEVL K DVYSFGIVLWELLTG LPY

MAP3Ks (Raf sub-group) that presented the characteristic motif GTxx(W/Y)MAPE

(123) C7 (60) C36(123) Consensus(123)

123

130

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236

IATLLRETLKALVYLHFHGHIHRDVKAGNILIDSNGAIKLADFGVSACMFDAGDRQRSRNTFVGTPCWMAPEVMQQLHGYDFKADIWSFGITALELAHGHAPFSKYPPMKVLLM IGSILKETLKALDYLHRQGHIHRDVKAGNILLDTNGVVKLADFGVSACMFDTGDRQRSRNTFVGTPCWMAPEVLQPGSGYNSKADIWSFGITALELAHGHAPFSKYPPMKVLLM IASILKETLKAL YLH GHIHRDVKAGNILIDSNG IKLADFGVSACMFD GDRQRSRNTFVGTPCWMAPEVLQ GY KADIWSFGITALELAHGHAPFSKYPPMKVLLM

Contigs MAP4Ks that presented the characteristic motif TFVGTPxMAPEV

Snf1: Alignment of part of the N-terminal catalytic domain and the T-loop domain of CitEST contigs and singletons encoding putative SnRKs, SNF1 and other SnRKs 18 contigs and two singletons

T-loop

Phylogenetic relationship between members of the SnRK families. Alignment of N-terminal catalytic and T-loop domains amino acid sequences was performed using AlignX (Vector NTI program).

Analysis of genes differently expressed:

MACROARRAY RT-PCRq

Category

Putative Function

Number of genes inGenes with differentialGenes with differential the membrane expression (%) expression (%) > 2x

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15

genes R

97

58,76 60 78,94 66,66 52,94 0 66,66 66,66 85,71 66,66 75 100 100 60 0

30,92 50 36,84 0 17,64 0 27,77 16,66 42,85 0 0 100 100 40 0

genes requeridos pelos genes R 10 MAPK MAP2K MAP3K MAP4K SNF1 NADPH oxidase SOD Nitrato redutase fenilalanina-amnia liase chorismate mutase isochorismate sintase PR-protenas canais de clcio 19 3 51 4 18 6 7 3 4 3 2 10 2

Category

Putative Function

Number of genes inGenes with differentialGenes with differential the membrane expression (%) expression (%) > 2x

C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26

Chalcone sintase Glutationa transferases Glutationa peroxidase peroxidases catalases canais de on-potssio metacaspases lipoxigenases cido cinmico 4-hidroxilase Epoxide hydrolase stress inducible protein

2 2 2 5 5 4 3 5 2 2 2

50 50 50 100 100 75 100 60 100 0 0

50 0 0 40 80 25 33,33 20 0 0 0

Genes de C. sinensis com expresso diferencial significativa 4,3 3,8 -log10(probt) 3,3 2,8 2,3 1,8 1,3 -4 -3 -2 -1 0 1 2 3 4 5 log2 das diferenas estimadas

Genes com expresso diferencial 100 90 80 70 Porcentagem 60 50 40 30 20 10 0

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10C11 C12 C13 C14 C15 C16C17 C18 C19 C20 C21 C22 C23C24 C25 C26

273 genes analyzed: 171 (62.63%) showed significant differential expression at the level of 5% probability

Categorias % do total de genes com expresso diferencial % dos genes com expresso diferencial > do que 2 vezes

Genes com expresso diferencial > do que 2 vezes 100 90 80 70 60 50 40 30 20 10 0

C 1 C 2 C 3 C 4 C 5 C 6 C 7 C 8 C 9 C 10 C 11 C 12 C 13 C 14 C 15 C 1 C6 17 C 18 C 19 C 20 C 21 C 22 C 2 C3 24 C 25 C 26

Of these 171 differential expression genes, 80 showed significant differential expression greater than 2 times, 38 of these genes were induced and 42 were repressed

Porcentagem

Categorias Induzidos Reprimidos

MACROARRAY VALIDATION - RT-PCRq

Macroarranjo RT-PCR

6 6 1 0 1 6 9 17 72 39 63 61 52 69 45 50 93 79 66 30 37 54 09 74 85 B T B A D P4 _5 O D3 A A A A A A A NP -B -A -B -C -C se -A -A 1ala 3K 3K 3K IPK M1 T1 se t N P P P HI ca RP ida SG KW MA MA MA rox AP pe M

9 genes analyzed: 5 genes induced in the macroarray were validated by RT-PCRq 3 of the 4 genes repressed in the macroarray were validated by RT-PCRq

Valores relativos de expresso diferencial

5 4 3 2 1 0 -1 -2 -3 -4 -5 -6

Genetic Transformation of Rangpur Lime (Citrus limonia Osbeck) with gene bO (bacterio-opsin) gene

Rangpur Lime rootstock represents 70% of the total cultivated area tolerance to citrus tristeza virus, water stress Low resistance to Phytophthora spp

pEL154

Methodology of Citrus Genetic transformation

PCR analysis

Southern Blot

Northern Blot

Protein immunoblot analysis of bO-His expressing 1: non-transformed tobacco plant 2: transgenic tobacco expressing His-tagged bO 3: non transformed Rangpur lime 4: Transgenic Rangpur lime line 2 5: Transgenic Rangpur lime line 1

Variations in size of lesions by Phytophthora nicotianae A: Non-transformed B: Transgenic line 2 C: Transgenic line 1

Acknowledgements: Francisco de Assis Alves Mouro Filho ESALQ/USP Beatriz M. J. Mendes CENA/USP Fernando A. Azevedo Centro Apta Citros Marcos Machado Centro Apta Citros Antonio de Goes UNESP/Jaboticabal Eric Lam Rutgers University Michael A. Lawton Rutgers University

Thank you
Helaine Carrer hecarrer@esalq.usp.br

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