Recent progress in the characterization of molecular determinantsin the Xanthomonas axonopodis pv. manihotis–cassava interaction

Valerie Verdier*, Silvia Restrepo, Gloria Mosquera, Veronique Jorge and Camilo LopezUMR5096, CNRS-Universite de Perpignan-Institut de Recherche pour le Developpement, 52 Av de Villeneuve,66860 Perpignan Cedex, France and Biotechnology Research Unit, Centro International de AgriculturaTropical (CIAT), AA 6713 Cali, Colombia (*author for correspondence; e-mail vverdier@univ-perp.fr)

Received 26 June 2003; accepted in revised form 2 April 2004

Key words: Cassava bacterial blight, cDNA-AFLP, detection, disease resistance, diversity, ESTs, geneexpression, QTL, resistance gene, RGC

Abstract

Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam), is a widespread diseasethat affects cassava (Manihot esculenta Crantz). Studies on the pathogen population structure, pathogendiagnosis, identification and expression of plant genes involved in resistance have been carried out. Dif-ferent molecular techniques were developed to assess the genetic diversity among the Xam populations.Characterization of Xam population dynamics over time had enable us to determine the different factorsthat are associated with resistance breakdown and those that influence the genetic structure or virulencephenotypes of the pathogen’s population. Methods for detecting the pathogen in vegetative plantingmaterials and true seeds were developed and contributed to reduce the impact of the disease. To betterunderstand the genetics of resistance a quantitative trait loci (QTLs) approach was developed. Using aPCR-based strategy with degenerate primers we isolated two resistance gene candidates in cassava. We alsocharacterized a region of a chromosome rich in R-gene like sequence. In this review we also report the mainresults obtained by transcript profiling methodologies, cDNA-AFLP and ESTs developed by the authors tocharacterize the genes involved in disease resistance. All together these techniques allowed the identificationof molecular markers either associated to CBB resistance or that may represent putative genes involved indisease resistance. This article reviews current knowledge on the molecular cassava–Xam interactions.

Introduction

Cassava bacterial blight (CBB) is a major disease,endemic in Latin America and Africa causingserious damage to cassava. The causal agent isXanthomonas axonopodis pv. manihotis (Xam).This vascular disease poses production concerns incassava, losses of between 12% and 100% affect-ing both yield and planting material have beenreported (Lozano, 1986). The amount of damagecaused by CBB varies throughout the world butcan be considerable (75% loss in Zaire in 1970)(Maraite, 1993).

X. axonopodis pv. manihotis is a foliar and avascular pathogen inducing a wide combination ofsymptoms, including angular leaf lesions, blight,wilt, stem exudates, stem canker and dieback(Figure 1). The infection process starts with theepiphytic multiplication of the pathogen, whichoccurs mainly close to stomata. Leaf penetrationtakes place through stomatal apertures or wounds.Xam is a vascular pathogen that enters vesselsafter a preliminary phase of intercellular develop-ment in the mesophyll. Host-pathogen interactionshave been well studied under controlled conditions(Verdier et al., 1990; Boher et al., 1995; Kpemoua

Plant Molecular Biology 56: 573–584, 2004.� 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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et al., 1996). Although a hypersensitive response(HR) is not observed in cassava leaves, data sug-gest that the vascular response of resistant varietiesto the pathogen can be assimilated to a putativevascular HR (Verdier et al., 1994; Kpemoua et al.,1996). Host-defense responses, associated with ahigh reduction of bacterial growth and progress inthe vascular tissues, have been well characterizedby Kpemoua et al. (1996). They include the pro-duction of phenolics, reinforcement of structuralbarriers (lignin, callose or suberin deposition), andvessel occlusion by pectic material and lignin-likecompounds (Boher et al., 1995; Kpemoua et al.,1996). When challenged by Xam, resistant plantsproduce particular digitate tyloses with anosmiophilic content that can be secreted within theinfected vessels (Kpemoua et al., 1996). A stronginteraction occurs betweenXam lipopolysaccharides(LPS) and cassava cell walls, suggesting that LPSmay be translocated from bacteria to the host cell,thus eliciting plant defense mechanisms (Boheret al., 1997).

The use of varietal resistance is the most real-istic and appropriate way to control the disease.The difference between resistant and susceptiblevarieties is expressed as a variation in the rate ofXam colonization of vascular tissues (Kpemouaet al., 1996). For this reason, resistance is usuallyconsidered as quantitative. Resistance to CBB hasbeen developed from Manihot esculenta and the

wild relative M. glaziovii, and is thought to bepolygenic and additively inherited (Hahn et al.,1979). However, in the past, genetics of resistanceto CBB have not been extensively studied incassava, and genes governing disease resistancehave been poorly documented for this crop.

Information on the pathogen’s populationstructure and geographic diversity is a prerequisitefor selecting disease resistance sources for cropbreeding. The population structure is determinedby the partitioning of genetic variation, and inconsequence the inference of population structureis also highly dependent upon the tools used toestimate genetic diversity (Leung et al., 1993).During the last decade, a wide range of techniqueshas been developed to assess the genetic diversityof Xam (Verdier et al., 1993; Restrepo and Verdier,1997; Restrepo et al., 1999b, 2000c). Different typesof RFLP probes were developed, either genomic,plasmidic or universal probes (ribotyping) (Verdieret al., 1993). The pathotype structure of thepopulation is of most direct interest to farmers,breeders and pathologists (Leung et al., 1993). Itconsists in the determination of the virulencespectrum of the strains on a set of differentialvarieties carrying different resistance factors.However, there are several limitations to pathotypeanalysis and it may not reflect the true geneticdiversity and evolutionary history of the isolatesexamined.

Figure 1. Field farmer showing symptoms of cassava bacterial blight caused by Xam, including angular leaf lesions, wilt, stem exudates

and dieback.

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A family of avirulence (avr) and pathogenicity(pth) genes has been identified in the genusXanthomonas (reviewed in Leach and White,1996). Members of the highly conserved avrBs3gene family encode proteins that contain a centraldomain composed of a near-perfect repeatedsequence of 34 amino acids (Leach and White,1996). This domain governs both specificity andvirulence (Yang and Gabriel, 1995). Othercharacteristics have been identified within avrBs3-homologs, such as presence of heptad repeatssimilar to leucine zippers and presence of threenuclear localization signal (NLS) sequences (Yangand Gabriel, 1995). An acidic transcriptionalactivation domain was identified in the carboxytermini of AvrBs3 family members and was shownto play a critical role for virulence activity (Yanget al., 2000). Recently, the type III secretion sys-tem-dependent translocation of the AvrBs3 pro-tein into the plant cell was demonstrated (Buttnerand Bonas, 2002; Szurek et al., 2002). Using animmunocytochemical approach, the authorsreported that AvrBs3 localizes to the nuclei ofinfected pepper leaves (Szurek et al., 2002). Dif-ferent hypotheses on the function of the AvrBs3members in eliciting plant defense response havebeen proposed (Leach and White, 1996; Bonas andVan den Ackerveken, 1997). Activities of avr genesare highly dependent on hrp genes (Van denAckerveken et al., 1996). AvrBs3 probably acts asa transcription factor as was demonstrated bytranscriptome analysis of susceptible pepper plant(Marois et al., 2002).

Although some non-pathogenic Xam strainshave been characterized genetically and pheno-typically, little is known about the epidemiologicalimportance of such strains in CBB disease (Rest-repo and Verdier, 1997; Gonzalez et al., 2002). Aswas recently observed in Colombia and in Africa,changes in the pathogen’s population structuremay lead to the overcoming of the deployedresistance (Restrepo et al., 2000c; Wydra andVerdier, 2002). We determine more accurately themolecular basis of pathogenicity of Xam strains bycloning and characterizing a gene named pthB. Itwas found to encode pathogenicity on the hostplant (Verdier et al., unpublished results). DNAsequencing shows that pthB is a new member ofthe Xanthomonas avrBs3 gene family (Bonas et al.,1989) harboring 12.5 internal 102-bp repeats. ThepthB gene presents striking features such as the

presence of nearly direct repeats each 1300-bplong, presence of perfect direct repeat 248-bp, andtwo perfect palindromic sequences. The complexstructure of pthB suggests a series of rearrange-ments and also that pthB may act as a transposon(Verdier et al., unpublished results).

Since bacterial blight is a major disease oncassava, we have carried out different studies onpathogen characterization and population struc-ture, epidemiology, disease diagnosis and on theidentification of genes involved in resistance. Herewe give a review on the recent advances inunderstanding mechanisms governing pathoge-nicity and resistance in the cassava–Xam interac-tions.

Characterization of the genetic diversity among

Xam strains

Determining best technique

Xam populations have been mostly characterizedusing RFLP (Berthier et al., 1993; Verdier et al.,1993). Nevertheless caution must be taken wheninferring phylogeny and pathogen populationstructures based on data from one or a fewmolecular markers (Nelson et al., 1994). A varietyof neutral markers should be used for character-izing the pathogen population structure (Leunget al., 1993). Others techniques (rep-PCR, ampli-fied fragment length polymorphism, AFLP) weretested for their usefulness to characterize Xampopulations (Restrepo et al., 1999b, 2000c). AFLPanalysis offer the possibility of a broader genomecoverage and its usefulness in characterizing bac-terial populations was shown by Janssen et al.(1996). Its applicability to the study of Xam pop-ulations and its high discriminatory power weredemonstrated by Restrepo et al. (1999b, 2000c).rep-PCR is simple, fast and a large number ofstrains can be analyzed. At the infra-pathovarlevel, rep-PCR is capable of discerning whetherpathovars represent a single or several evolution-ary lines (Louws et al., 1995), but in the case ofXam, rep-PCR did not allowed the differentiationamong RFLP haplotypes. Our results showed thatthe pthB gene of Xam is the best target for phy-logeny purposes and for relating pathogenicitygene function with genetic variability. RFLP usingthe pathogenicity gene pthB as a probe, was the

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most informative and discriminative technique forassessing the genetic diversity of Xam (Restrepoet al., 1999a, 2000c) and therefore is the techniquethat we should recommend for further analysis.

Xam populations: a high potential to change

The results obtained from our studies permitted tounderstand how diversity is distributed in spaceand how fast a Xam population can evolve andwhat factors contribute to the change. Consider-able variation has been observed among Xamstrains from South America but was limited fromother regions. In Africa, the first results obtainedsuggested a clonal population structure for thispathogen (Verdier et al., 1993). However, in morerecent studies, African strains had revealed higherlevels of diversity (Assigbetse et al., 1998; Verdieret al., unpublished results). Recent analysis of 218African strains collected in Togo allowed the dis-tinction of nine different haplotypes (Verdier et al.,unpublished results). From 1995 to 2000, variousprospections have been conducted resulting in thecollection of 906 isolates from Colombia, Vene-zuela and Brazil (CIO collection available atCIAT, Biotechnology unit). All the isolates havebeen characterized resulting in the description of111 haplotypes (Restrepo and Verdier 1997;Restrepo et al., 2000c, 2004). In Brazil, 79 strainscollected between 1941 and 1996 from three ed-aphoclimatic zones (ECZs) were analyzed byRFLP/pthB leading to the identification of 38haplotypes (Restrepo et al., 1999a). A geographi-cal differentiation of Xam strains was demon-strated. Compared to the Xam population fromColombia and Venezuela, Brazilian populationwas the most diverse. Considerable genetic diver-sity was also revealed in Venezuela. Twenty-eightpthB haplotypes were found among the 91 isolatescollected (Verdier et al., 1998a). The 28 haplotypeswere clearly distinct from the haplotypes previ-ously described for Brazilian and Colombian Xamcollections. In Colombia, from 1995 to 1999, 45different RFLP types or haplotypes were detectedamong 736 strains (Restrepo et al., 2000c, 2004).RFLP clustering were very similar using eithergenomic or plasmid probes, supporting the hypoth-esis of co-evolution between plasmid and chromo-somal genomes (Restrepo and Verdier, 1997).

The genetic diversity of strains was studied atdifferent levels (hierarchically) in space and over

time in Colombia (Restrepo et al., 2000c). Thescreening for RFLP variation in the Xam popu-lations indicated that a high level of variation atthe DNA level has remained constant over time.Xam populations are unstable and changes occurrapidly, in less than a year. Also during a cropcycle a new population structure is established. Wedemonstrated the existence of a geographical dif-ferentiation of the Xam strains in different ECZs.Our analysis also permitted to understand thefactors that shape the population structure. Firstthe deployment and widespread use of resistantgenotypes accelerate the selection of new variantsin the populations. Also the exchange of contam-inated cassava materials contributes to themigrations of strains and consequently influencesthe genetic structure of Xam populations. Finallychanges in environmental factors (rainfall andtemperature) also influence the genetic diversityand population structure of Xam. All this infor-mation has been very useful in designing breedingstrategies. For example we recommend evaluatinggermplasm with a specific set of Xam strains thatrepresents the range of genetic diversity within anecozone. Considering the variability of the popu-lations and the migrations, improving quarantineprecautions are recommended to prevent impor-tation of infected planting materials to areas withlow disease incidence.

Identification of pathotypes

In a first survey, 26 RFLP/pthB haplotypes weredescribed within Xam populations in Colombia(Restrepo and Verdier, 1997). These results per-mitted the selection of a set of 26 Xam strains forinoculation on 17 potential cassava differentialspreviously characterized (Sanchez et al., 1999;Restrepo et al., 2000b). A cultivar · strain inter-action was shown to be highly significant and theexistence of pathotypes was clearly demonstrated(Restrepo et al., 2000a). One pathotype was de-scribed for ECZ5, 5 for ECZ1 and 5 for ECZ2 inColombia (Restrepo et al., 2000a). The set of hostdifferentials was further extended to other varietiesfrom ECZ5 and 2–4 and pathotype evaluationsincluded new ECZ2–4 strains (Restrepo et al.,2000c). The latest results we obtained indicate theexistence of 9 pathotypes in ECZ1, 13 pathotypesin ECZ2, 4 pathotypes in ECZ5 and 3 pathotypes

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in ECZ2–4 (Restrepo et al., 2004). By studying thetemporal dynamics of the Xam population struc-ture we have shown that changes in the haplotypiccomposition reflects changes in the pathotypiccomposition. Based on this, we recommend toscreen cassava cultivars for resistance to CBB bymonitoring the changes in the population usingRFLP/pthB and identifying the pathotypes byselecting the strains among the new haplotypescharacterized. Varieties should be screened withnew pathotypes specific to each ECZ. Followingthe same strategy, pathotypes were also identifiedin Brazil (Restrepo and Verdier, unpublishedresults), Venezuela (Verdier et al., 1998a, b) andAfrica (Wydra et al., unpublished results). It willbe interesting to know how molecular diversity orchanges in the pthB gene structure of Xam mayaccount for resulting pathotype. Changes in theavr/pth gene structure that cause a variation invirulence pattern of the strain can be due to singlemutations at the 3¢ terminus of the avr alleles as inthe case of X. oryzae pv. oryzae (Vera Cruz et al.,2000). One strategy could be to sequence thecopies of the plasmid-borne pthB gene in the newpathotypes. Also it could be interesting to deter-mine if some of these pthB copies have a measur-able fitness function in Xam. This could beachieved by mutating individual pthB gene copiesand by testing the pathogenicity effect.

Identification of specific markers related

to pathogenicity

We attempt to characterize genes putatively in-volved in pathogenicity. Developments in DNAtechnology have provided new tools for identifyingpotentially useful markers to aid in understandinghost-pathogen interactions. A selected set ofpathogenic and non-pathogenic Xam strains fromour collection was analyzed using PCR-basedtechniques (Gonzalez et al., 2002). Different pri-mer combinations were used including primersspecific to the Xam pathogenicity gene (pthB)along with RAPD or AFLP primers. PCR tech-niques using primers corresponding to the patho-genicity gene, pthB allowed distinction betweenpairs of pathogenic and non-pathogenic strainsand permitted the isolation of DNA fragmentsspecific to pathogenic strains of Xam. Polymorphicsequences between pathogenic and non-pathogenic

strains were classified into different groups.Sequence analysis showed significant homologywith bacterial pathogenicity island, genes involvedin pathogenic fitness and regulators of virulence(Gonzalez et al., 2002). We found similarities toseveral positive transcriptional regulators, a simi-larity to a TonB dependent receptor, and severalsimilarities to bacterial effector proteins (Gonzalezet al., 2002). We also confirm previous workshowing that sequences related to the avrBs3 genefamily (pthB) are conserved among pathogenic andnon-pathogenic Xam strains and that they mayplay a role in Xam pathogenicity. We demon-strated the high-resolution power of new PCR-based techniques in differentiating highly relatedbacterial strains. Further studies are needed inorder to investigate whether the different frag-ments generating polymorphism correspond togenes related to pathogenicity.

Detecting the pathogen for a better management

of the disease

The movement of infected plants is a major meansof Xam dispersal. A reliable and sensitive diag-nostic procedure is necessary for the safe move-ment of cassava planting material. We have usedthe sequenced pthB gene of Xam to set up differenttechniques for detecting the pathogen. Wedesigned primers that did not show any similaritywith known sequences in data banks. This DNAsequence provided primers specific for Xam andallowed the amplication of a 898 bp fragment inall Xam strains tested (Verdier et al., 1998b). Thetest is highly specific as no DNA fragments wereamplified from the 46 other bacterial strains,including six unidentified epiphytes. The PCRprocedure is a rapid method that can be easilyadapted in any place for screening of suggestive orinfected plant tissue without prior bacterial isola-tion or DNA extraction. In practice, preparingtissue extracts suitable for PCR require less than1 min per sample. The PCR method allowsdetecting three viable cells per reaction. The PCRwas then improved by designing internal primersfor the 898 fragment that were used in anested-PCR assay (Ojeda and Verdier, 2000). Thenested-PCR increases sensitivity of detection andpermitted to detect successfully the pathogen inseeds. The seed-washing procedure is non-destructive,

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and the bacterium DNA does not have to beextracted before processing the seeds, allowing thematerial to be tested in only one day. In the case ofexchanges of cassava germplasm through trueseeds or material propagated in vitro, the PCR andnested-PCR procedures are simple and fast, andcan be easily implemented to certify plant materi-als (Guevara et al., 1998).

The dot-blotting procedure that we havedeveloped uses a DNA fragment from the pthBsequence data, which appears to be a pathovar-specific DNA probe. It is a simple and specificmethod for detecting Xam directly in cassava planttissues (leaves, stems, fruits, seeds and embryos)and for testing colonies of putative isolates of Xam(Verdier et al., 1998b). Sensitivity is 100 to 10times less than PCR but the dot-blot technique issensitive and rapid and permits large-scale testingof cuttings for culture indexing at relatively lowcosts and little equipment (Verdier and Mosquera,1999). The different detection methods should beadaptable to the specific needs of each laboratoryand will depend on the available facilities.

QTL analysis of CBB resistance: a complex

interaction between the plant and the pathogen

Cassava is an allogamous highly heterozygousspecies and suffers from inbreeding depressionmaking difficult classical genetic studies currentlyused with inbred lines. F1 population from crossesbetween non-inbred parents is the material usedfor the construction of the first cassava geneticmaps through double pseudo-testcross strategy(Fregene et al., 1997). These maps allow dissectionof complex trait through quantitative trait loci(QTL) analysis and are of great interest for markerassisted selection (MAS).

CBB resistance is considered as quantitativeand therefore a QTL based approach has beendeveloped to better understand its genetic control(Jorge et al., 2000, 2001). Segregation analysis hasbeen conducted in the intraspecific F1 cross used toconstruct the original map (Fregene et al., 1997).An half-sib BC1 population of 243 individuals wasdeveloped by backcrossing five F1 genotypes withthe female parent to detect recessive allele ofresistance genes (Jorge et al., 2000, 2001; Zinsouet al., unpublished data). We evaluated CBBresistance in F1 genotypes in replicated trials in the

field under high disease pressure over three con-secutive crop cycles. Resistance was also evaluatedunder controlled conditions by inoculating twosegregating populations, F1 and BC1 with differentstrains of Xam representative of the geneticdiversity found in their respective ECZs.

Our results confirmed the quantitative natureof resistance to CBB (Table 1). Although parentsof the segregating populations studied were bothtolerant to CBB, quantitative resistance is segre-gating in F1 and BC1 progenies. A particularlyinteresting result is the specificity of CBB quanti-tative resistance, as was shown by greenhouseexperiments with some Xam strains (table 1).Changes in QTLs from crop to crop cycle weredetected in field trials. The pathogen population(Xam) was monitored over the period both bymolecular and pathogenic assays and changes inQTL detection over the 2 years could be correlatedwith changes in pathogen population structure(Jorge et al., 2001). Three QTLs located in linkagegroup D, were conserved over the two crop cyclesin field and also in greenhouse evaluations(table 1). They appear to be an introgression of M.glaziovii. Most of the QTLs involved in resistanceto African strains were different from those cor-responding to Colombian strains (Zinsou et al.,unpublished data). This result confirm the QTLspecificity found on the F1 population analysis(Jorge et al., 2000) and are of special interest forQTL identification and breeding strategies forCBB resistance in Africa and Colombia. First thehigh specificity of disease resistance and second thehigh level of pathogen diversity in Colombiacompared to the relatively low level in Africa makethe breeding strategies different in the respectivecountry. In Africa, we were able to evaluateresistance and to identify QTLs in greenhousechoosing a limited number of Xam strains. Bycontrast, in Colombia, as pathogen populationsare highly diversified and structured by ecozones,field resistance evaluation is highly recommendedthrough the identification of ecozones specificQTLs. In conclusion, these studies identifiedmolecular markers useful for MAS, a techniquethat can accelerate the long, multiple-season pro-cess of breeding for CBB resistance and othercharacteristics. For example, linkage group D isalso known to be involved in ACMV resistanceand other agronomic traits like dry matter content.Using specific alleles (female allele) of markers of

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this group, we may select early F1 clones bearingtheses favourable alleles (bands) for CBB resis-tance and other important traits.

Towards the characterization of resistance

genes in cassava

Some plant pathogen interactions are governed byspecific interactions between pathogen avr geneproduct and the product of plant disease resistancegenes (R-genes) (Dangl and Jones, 2001). Thisinteraction can occur directly (receptor-ligandmodel) as demonstrated in the AVR–Pita interac-tion (Jia et al., 2000). Recently an indirect per-ception of pathogen derived proteins within a

complex has been described leading to the guardmodel concept (Mackey et al., 2002). The mostprevalent class of R-genes in plant genomesencodes NBS-LRR-containing proteins that caninclude or not a TIR domain at their N-terminus.The well-conserved regions of the NBS domainshave been used to isolate putative R-genesequences or resistance-gene candidates (RGCs) indifferent plant species using a PCR-based strategywith degenerate primers (Meyers et al., 1999).Most of the RGCs colocalized with R-gene locipreviously identified and/or QTL associated toresistance to pathogens. We developed a similarapproach to search for candidate R-genes in cas-sava using degenerate primers targeted to TIR andNBS regions (Lopez et al., 2003). Two TIR-RGCs

Table 1. QTLs related to CBB resistance detected in cassava.

Name of QTLsa Linkage groupb Most significant markersb Resistance in greenhousec Resistance in field

XM1 D (female) GY222, GY167 CIO-84, ORSTX-27 Field

XM2 L (female) GY141 CIO-84, ORSTX-27

XM3 N (female) AD4b CIO-1, Uganda12

XM4 N (female) GY145 CIO-1, Uganda12

XM5 X (female) CBB1 CIO-136

XM6 B (female) GY147 CIO-295

XM7 D (female) GY179 ORSTX-27 Field

XM8 D (female) O19 ORSTX-27 Field

XM9 C (male) GY54, SSRY93 CIO-84, Uganda12

XM10 G (male) GY93 CIO-84

XMF2 E (male) CDY123a Field

XMF3 F (female) GY186 Field

XMF4 I (female) AC1 Field

XMF5 M (male) GY67 Field

XMF6 B (male) GY72 Field

XMF7 E (female) GY205 Field

XMF8 A (male) X17 Field

XMF9 N (female) GY124 Field

XMB1 N (female) GY218 CIO-84, CIO-136

XMB3 M (female) GY192 CIO-295

XMB4 H (female) SSRY52 ORSTX-27

XMB5 nnd (male) SSRY148 CIO-295

QTL1 O (female) SSRY6 GSBP2507, GSBP2511

QTL2 O (female) SSRY170 GSBP2511, ORSTX-27

QTL5 A (male) GY12 GSBP2507, Uganda12

QTL6 nnd SSRY83 GSBP2506

aAccording to Jorge, 2000; Jorge et al., 2000, 2001, XM, XMF: identified through F1 population analysis; XMB, QTL: identifiedthrough BC1 population analysis.bAccording to Fregene et al., 1997; Mba et al., 2001.cResistance was evaluated by inoculating Xam strains. QTL detected correspond to each mentioned strain (i.e., XM1 is conferringresistance to CIO84, ORSTX27 and in the field).dNot named.

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and ten NBS-RGCs classes were identified. Fur-ther sequence analyses permitted to group theNBS-RGCs sequences into two subfamilies thatwere predicted to contain or not a TIR domain.Southern blot and BAC screening analyses showedthat all RGCs are found in single or low numbercopies, except one (RCa6) that represents a largeand diverse gene family. The partial sequence of aBAC clone (11E5) obtained with the RCa6, andcontaining five NBS sequences, permitted theisolation of two full-length RGCs (Lopez et al.,2003). At least one of them is constitutivelyexpressed in cassava tissues as shown by RT-PCR.Two RGC-containing BACs were mapped onlinkage group E and four RGC-containing BACswere mapped in the distal region of the linkagegroup J (Figure 2). In this region a cluster con-taining at least 14 NBS sequences was identified(Figure 2). No association between RGCs andQTLs controlling disease resistance was found butso far few resistances genes or QTLs have beenmapped in cassava. More phenotypic data relatedto disease resistance is required in order to estab-lish the linkage between RGCs and R-loci and toconduct further mapping analysis. Identification ofRGCs in cassava provided new valuable tools forMAS. These tools can be used in high-resolutiongenetic mapping as an effective tool in map-basedcloning of R-genes in cassava.

Gene expression analysis by transcript profiling

and ESTs analysis: new resources for better

understanding the cassava–Xam interaction

Some studies have been conducted to identifydifferentially expressed genes in cassava tissueinfected with Xam. In particular, a- and b-esterasesand a peroxidase have been shown to be usefulmarkers of pathogenesis with Xam (Pereira et al.,2001, 2003).

In our laboratory, high throughput methods oftranscript profiling have been developed to studycassava–Xam interactions and to annotate anddetermine gene functions. The use of cDNA-AFLP has been described as an extremely efficient,reproducible and sensitive method for isolation ofdifferentially expressed genes (Bachem et al., 1996;Cooper, 2001). cDNA-AFLP was used to identifycassava genes expressed during infection with Xamby comparing the differential gene expression of acompatible (susceptible plant) and incompatible(resistant plant) reaction over time (Santaellaet al., 2004). Transcript-derived fragments (TDFs)were selected when their expression was up-regu-lated during the bacterial infection in the resistantvariety. Of �3600 cDNA fragments inspected inthe resistant genotype, approximately 9% were up-regulated, indicating that they may be involved inthe defense response mechanisms. The DNA

Dist MarkercM Name

SSRY172 13.1

rCDY76 4.4 CDY74 4.4 rAD1c 10.7

J9c 10.8

E14b

16.2

rK2a

24.5

rGY34 8.7

rSSRY101 6.6

rSSRY50.7 rSSRY212.9 rSSRY229

18.7

BAC 11E59.7

BAC 80P22 106C128.4 BAC 75D2

BAC106C12

BAC11E5 BAC80P22BAC75D2

BAC68L3 BAC89H13

Figure 2. Schematic map of the genetic linkage group J showing the position of the RGCs candidate-containing BACs. Blue boxes

indicate the NBS sequences.

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fragments of interest were excised from gels,amplified, cloned and sequenced. Sequence analy-sis shows similarities to defense related proteinsthat are associated with plant defense in otherplants. Putative products of other genes identifiedhere may play a role in programmed cell death,regulation of gene transcription and signal trans-duction. Reverse transcription PCR was used toconfirm differential expression of some TDFs andin most cases shows a good correlation with theresults obtained by cDNA-AFLP (Santaella et al.,2004).

The expressed sequence tags (ESTs) derivedfrom single pass sequencing of cDNA provide aninvaluable resource for the identification of genesassociated with specific conditions. We generated abroad collection of EST targeting two economi-cally important characters, starch content and CBBresistance (see Anderson et al., this volume). A5700 cassava unigene set was characterized (see thisvolume). Some of these genes show similarity togenes involved in defense mechanisms, homologsto R proteins, chitinase and receptor like proteinkinases. Data generated through our studies ontranscript profiling and by others (Pereira et al.,2001, 2003) will permit unraveling the complexpathway signal in the cassava–Xam interaction.

Conclusions and prospects

Bacterial blight is an important cassava diseaseand various studies have been carried out to ex-plore the genetic diversity and population struc-ture of Xam and its implication on host resistance.From the above review we can see that significantprogress have been made both on the pathogenside and on the plant side. Apart from the pthBpathogenicity gene previously identified (Verdieret al., unpublished results), other new Xam genesputatively involved in pathogenicity were identi-fied. However, multiple bacterial genes are in-volved in plant pathogen interactions.Traditionally, transposon mutagenesis has beenused as a method of choice to identify genes in-volved in pathogenicity (Mills, 1985 and referencestherein). This technique has yielded importantinformation about a number of genes involved indisease development in pathogens as Pseudomonassyringae or Erwinia. carotovora (Mills, 1985).However, ‘hot-spots’ for transposon insertion,

secondary transposition with possible genomerearrangements, low throughput and the genera-tion of lethal mutations limit the use of thismethod (Mills, 1985). Many subtle interactionsbetween the pathogen and its host may be lostwhen the pathogen is disabled by mutagenesis(Dellagi et al., 2000). An alternative approach is toinvestigate changes in gene expression during theinteraction. At the present high-throughput tech-niques are available to characterize the expressionof thousands of genes in a single experiment. Thefirst plant pathogen microarray was used to iden-tify genes in Erwinia chrysanthemi that are regu-lated in the host plant (Okinaka et al., 2002).DNA microarray is a useful tool that could allowgene expression analysis even in non-sequencedgenome such as Xam. The construction andexploitation ofXammicroarrays is in progress in ourlab and will help in identifying the genes that alongwith pthB are important in bacterial virulence.Microarrays can also be exploited and applied to theanalysis of DNA polymorphisms. The method iscalled Diversity Array Technology (DArT�)(Jaccoud et al., 2001). Using this method, monitoringof Xam populations diversity will be facilitated.

Determining the exact function of the genesinvolved in bacterial virulence is difficult withoutknowing the host plant genes that are involved inresistance. Although genes governing resistance toXam in cassava have not yet been identified, somegood candidates have been characterized either bya QTL approach or by developing transcript pro-filing methodologies. Further studies confirmingthe expression of candidates genes in the plantchallenged with Xam are needed. Another alter-native is to use the yeast two-hybrid system that isa method of choice to fish for resistance genes.This technique was developed for studying theinteraction of AvrPto and Pto (Scofield et al.,1996), and of Pto and other genes acting down-stream of the phosphorylation cascade (Zhouet al., 1995). It is possible to use the pthB gene asbait in the yeast two-hybrid system to isolate thecorresponding interacting genes from plant cDNAlibraries and to determine whether these genes areinvolved in controlling host-defense responses.

The different methodologies developed by ourgroup provide a major resource for studying cas-sava biology and in particular, cassava resistancegenes to important pathogens. The EST collectionthat we developed is promising in unraveling the

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complexities in cassava gene expression. We alsoprovide a resource for comparative genomics.Additional efforts are needed in order to furtheridentify key genes involved in CBB resistance butalso other genes of interest to the cassava commu-nity. The generation of a large collection of ESTs isthe first step towards the generation of microarraysfor further cassava gene expression studies.

To validate the function of the RGCs and otherputative R-genes it is necessary to have a robustand reproducible genetic transformation protocol.The first genetically transformed cassava plantshave been produced in 1996 and since the tech-nique has been expanded to other cassava cultivars(Schopke et al., 1996; see also in this volume). Thetechnology is highly reproducible and allows theinsertion of genes of agronomic interest. Otherstrategies such as RNA interference could also beconsidered and will permit a large-scale functionalscreening. When suitable viral vector becomeavailable for cassava, virus induced gene silencing(VIGS) (Lu et al., 2003) might also become amethod of choice to validate the candidates genethat we already have identified or those that arebeing under process of identification once the ESTdatabase will be fully exploited.

Acknowledgements

We gratefully acknowledgeW. Roca, J. Tohme, M.Bonierbale, C. Iglesias, M. Fregene, A. Ghesquiereand M. Delseny and for their support. Thanks toK. Assigbetse, M. Chavez, M.C. Duque, C.Gonzalez, S. Ojeda, M. Santaella, E. Suarez, C.Velez, P. Zuluaga for their significant contributionto the work. Our research was supported by grantsfrom the European Community (project INCO-DC contract no. IC 18CT 980306), from InstitutoColombiano para el desarrollo de la ciencia y latecnologıa ‘Francisco Jose de Caldas’ (COL-CIENCIAS) (contract no. 344-98, code 2236-12-051-98) and ECOS-Nord (Action C03A02).

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