Jo K.Tugume*,Geoffrey Tusiime,Alln Mle Sekmte,Roin Buruchr,Clre Mugish Muknkusi

aMakerere University,Department of Agricultural Production,Kampala,Uganda

bInternational Centre for Tropical Agriculture(CIAT)/Pan African Bean Research Alliance(PABRA),Kampala,Uganda

Keywords:Xanthomonas axonopodis Interaction Gene expression Diversity

ABSTRACT Common bacterial blight(CBB)is associated with common bean(Phaseolus vulgaris L.),an important grain legume for human consumption worldwide.The disease,caused by Xanthomonas spp.is spread mainly through seed.This paper focuses on the diversity of X.axonopodis pv.phaseoli and X.fuscans subsp.fuscans and interactions between related bacteria and the bean host.Review has suggested that the diversity and taxonomic studies of these pathogens are not exhaustive,especially in areas where detailed molecular analysis has not been conducted and previous characterizations were based on phenotypic features and PCR-based techniques.Also,no study has confirmed differential pathogenicity on bean genotypes based on compatible versus incompatible reactions.However,isolates react differently to wild and domesticated bean sources of resistance in common bean genetic backgrounds.A systematic approach will be required to investigate global changes in gene expression among different sources of resistance in a common bean background.The bacterial isolates that cause CBB should be functionally characterized using genotypes containing major quantitative trait loci(QTL)for CBB resistance.These studies will increase understanding of resistance and how it is manipulated by pathogens.

1.Introduction

Common bean(Phaseolus vulgaris L.)is a major food legume grown worldwide.It is an important crop in the entire Great Lakes region of Africa for food as well as income security[1].Common bacterial blight disease(CBB)is a major constraint to bean production worldwide[2-4].Infection by bacteria occurs largely through stomata,colonizing mesophyll cells and causing leaf spots and sometimes chlorosis[5].Bacteria can be spread through seed and in the field by rainsplash[6].Boersma et al.[2]reported a yield reduction of up to 35%in susceptible varieties of beans;however,losses＞40%have been observed under conditions favorable for the disease[3].Opio et al.[7]reported a reduction in yield of 11.5 kg ha-1at growth stage R7 corresponding to a 1%increase in the number of leaves infected with CBB.The disease is a principal constraint in mid-altitude production areas and is favored by warm temperatures and high relative humidity[4,8].This paper reviews the literature on common bacterial blight disease-causing pathogens and their interaction with P.vulgar is,in order to summarize the current knowledge of the mechanisms of pathogenicity and host resistance.The diversity of common bacterial pathogens,host responses,and associated genes for resistance is reviewed.This information will increase understanding of resistance in P.vulgar is and will assist breeding programs in developing beans with durable resistance against common bacterial blight.

2.Diversity of CBB pathogens infecting P.vulgaris

Common bacterial blight pathogens belong to the genus Xanthomonas,a Gram-negative group of γ-proteobacteria.Since the identification of Xanthomonas as a causal agent of CBB in1897,the taxonomy of infecting strains has been debated,owing to their changing genetic diversity even in a common bean host[9].Until 1995,fuscous and non-fuscous strains were grouped in a single taxon,X.campestris pv.phaseoli[10].Following taxonomical revision of the genus Xanthomonas,pathovar phaseoli was transferred to X.axonopodis,with fuscous strains forming a variant within this pathovar[10].The nomenclature of fuscous strains within X.axonopodis was again changed to X.fuscans subsp.fuscans(Xff)[8].Using a suppression subtractive hybridization approach,Alavietal.[11]grouped a world collection of CBB pathogens into X.fuscans subsp.fuscans and three genetic lineages,1,2,and 3,belonging to X.axonopodis pv.phaseoli(Xap).That study revealed further diversity within the Xap group.Although lineages 2 and 3 were closely related,lineage 1 was distinct and encoded a putative type III secretion system(T3SS)containing a salmonella pathogenicity island(SP-1)that could lead to its classification into a subspecies.In contrast,Zamani et al.[12]and Lopez et al.[13],using PCR-based methods,found limited diversity in the Iranian and Spanish isolates of Xap that are more predominant in those countries.Duncan et al.[14]later reported that brown pigmentation is not a phenotypic feature confined to the Xff strain,as some X apisolates were also found to produce brown pigment.This finding means that brown pigmentation cannot be relied on as a phenotypic feature for classifying CBB pathogens.Recently,Aritua et al.[15]suggested that CBB pathogens could be further divided into Xap, Xff and Lablab strains using multiple-locus sequence analysis.However,this grouping is likely to change as more data are generated by deep sequencing and other platforms. Constantin et al. [16] has already suggested revisions to classify fuscous strains as X.citripv.fuscans and X.phaseoli pv.phaseoli.In this review,the previous nomenclature of X.fuscans subsp.fuscans and X.axonopodis pv.phaseoli is maintained.

3.Virulence factors in CBB pathogens of beans

Successful infection of a plant results from the actions of multiple virulence factors acting together to promote disease.Differences in virulence factors(effectors)among isolates are informative about the pathogenicity mechanisms that usually determine the host range and aggressiveness[17].Through genome sequencing of Xanthomonas strains isolated from P.vulgaris,six secretion systems and several effectors have been revealed,but they are not common to all infecting strains[11].These multiple virulence factors produced by different secretion system have been reported to be present in CBB-causing pathogens.Alavi et al.[11]found a unique T3SS in the genetic lineage GL1 of Xap composed of hrp2 and salmonella pathogenicity island that was not found in Xff and other lineages.Aritua et al.[15]found 10 effectors conserved in 26 sequenced isolates of Xap,Xff,and Lab-labstrains (XopR,XopV,XopE1,XopN,XopQ,XopAK,XopA,XopL,AvrBs2,and XopX).However,some unique effectors were also found specific to Xap(XopC2),Xff(XopF2),and Lab-lab(XopA1).The roles of these strain-specific effectors in determining virulence and host specificity remain to be identified.Bacterial pathogens normally use effectors to modify host responses to create favorable conditions for their own survival[18].In some pathosystems,some effectors have been found to affect photosynthesis[18]and translocation of photosynthates[19],while in others they have been found to affect the permeability of cell walls,thus allowing nutrient leakage to the pathogen[20].The role of unique virulence factors in the infection process can be examined by study of their expression patterns during the infection process.

4.Differential response of common bean genotypes to CBB pathogens of beans

Although variation in virulence has been observed among CBB isolates,differential pathogenicity on different common bean genotypes based on compatible versus incompatible reactions has not been confirmed[14,21-23].Interaction of P.vulgaris with CBB pathogens has been shown largely not to follow the gene-for-gene model[22,24].However,gene-for-gene interactions were observed in tepary bean(P.acutifolius A.Gray),a close relative of common bean[22].It was further observed that this gene for gene interaction is non-specific in the P.vulgaris background.In Ugandan and Ethiopian isolates,Opio et al.[22]observed eight distinct CBB responses on seven tepary bean genotypes(Table 1)and 90%of the tested isolates induced symptoms on each of the 20 bean genotypes tested.In South Africa,Fourie et al.[25]observed that the pathogenicity of all but one isolate was similar to that of race 2,implying limited CBB pathogen diversity.However,variation in the severity of symptoms among the tested genotypes implied broad resistance.Vandemark et al.[26]observed that introgressed resistance from the wild species using markers BC420 and SU91 followed a recessive epistasis model,in which the function of corresponding QTL are modified by actions of other genes in the bean genome.Whereas plants with the BC420//BC420/su91//su91 genotype were susceptible,those with SU91//SU91 and SU91//su91 showed an intermediate response when homozygous for bc420.Thus,the BC420 QTL on its own appears to have no effect on disease response in plants with su91//su91.

Breeding programs over the years have introgressed CBB resistance in different market classes,including Andean accessions,which are generally more susceptible than Mesoamerican accessions.Mutlu et al.[27]developed an ABCP-8 genotype using resistance markers SAP6 and SU91 that showed greater resistance to common bacterial blight,with 6%infection in field and greenhouse tests,than the recurrent parent Chase(33%field and 46%greenhouse)and a susceptible check Othello(59%field and 100%greenhouse).Using the same markers,Miklas et al.[28-30]developed USDK-CBB-15,a dark red kidney,USWK-CBB-17(a white kidney),and USCRCBB-20(a cranberry)that had disease scores of 3.6(the most resistant),4.8,and 5.3.Variation in resistance in developed cultivars can be attributed to different backgrounds used in the crosses and differences in pathogen aggressiveness.

Functional characterization of CBB isolates from different regions of the world has shown no single source of resistance that is effective in a common bean background against all isolates[14,21].The effectiveness of CBB resistance varies widely with the aggressiveness of the pathogen[25,31].The high resistance observed in VAX 3 and VAX 6 inoculated with the less aggressive isolate-ARX8A became intermediate on exposure to the more aggressive isolate Xcp25[31].Resistant sources containing the marker SAP6 are thus effective against less aggressive isolates[14,21,31].VAX 4,in comparison,contains SAP6-and SU91-linked QTL and is the most resistant bean line against a range of CBB isolates except for isolate 96-05 from Honduras,which can overcome all known major sources of resistance to CBB[21](Table 2).Duncan et al.[14]also reported that VAX lines 3-6 were effective against a wide range of CBB isolates.Whereas some isolates can overcome resistance that originates from common bean(SAP6-linked resistance),other isolates tend to overcome resistance originating in the wild(SU91+BC420)[14,21].Whereas some studies have reported New World isolates to be more virulent[14],other studies have shown Old World and African isolates to be more virulent[19,21,22].The specificity of different sources of resistance to CBB isolates suggests that someisolates co-evolved with wild beans and others with domesticated beans.Vandemark et al.[32]suggested that there was specificity of SAP6-linked resistance to isolates of CBB.Although SAP6 and Xa.114-linked resistance provide some level of pod resistance,other identified resistance linked markers are ineffective[23].It is important to note that genes involved in CBB resistance in pods are different from those for foliar resistance[33].Wild sources of resistance like SU91 and BC420 had previously been reported to carry a yield penalty,though a recent study by Miklas et al.[3]suggests otherwise(Table 3).In that study,plants homozygous for BC420//SU91 yielded better than plants homozygous for bc420//su91 in a non-disease environment.This finding suggests that BC420 could be linked to genes that enhance yield.Thus,durable resistance can best be achieved by careful combination of wild and domesticated gene sources[14,21].

Table 1-Interaction of isolates of Xanthomonas spp.with seven genotypes of tepary bean,P.acutifolius.(Adapted from Opio et al.[22].)

Table 2-Differential response of CBB pathogens to selected resistance QTL in different genetic backgrounds.

5.Identifying individual genes for resistance to CBB

Identifying or mining individual CBB resistance genes among quantitative trait loci(QTL)is in progress,and accordingly limited information is available for annotation of defense associated genes.Using Medicago truncatula Gaertn.as a model species,21 genes in the BAC clone AK7 were predicted for BC420-linked QTL[34].However,of these only six genes were found in common bean expressed sequence tags(ESTs)and only three in P.coccineus ESTs.Shi et al.[34]annotated QTL linked to SU91 from the BAC clone 32H6 using Medicago spp.as the reference genome and predicted 16 genes.None of thesegenes was present in the EST database for P.vulgaris and P.coccineus.One of the 16 genes associated with SU91 belongs to the UDP-glycosyltransferase(UGT)family,which plays a role in ascorbic acid homeostasis,ABA metabolism,and resistance to hemi biotrophic pathogens such as Xanthomonas spp.of beans[35].However,these 16 genes are conserved in the G19833 susceptible and OAC Rex resistant genotypes,suggesting a reduced role in resistance[36].Sequencing of genomic library clones derived from Chr.04 of the bean genotype OAC Rex revealed10genes with similarity to Chr.06 and Chr.08 of common beans[37],but the linkage between the genes and the CBB resistance markers BC420 and SU91 was weak.The only four genes that were defense-related lacked the key functional motifs(CC,NBS,TIR,kinase,and transmembrane domain.Most genes identified by Cooper[37]were associated with transposable elements,suggesting that they are not stable in the bean genome.Other genes not associated with transposable elements included those encoding L-ascorbate oxidase,callose synthase,and a hypothetical protein.Similarly,Zhu et al.[38]annotated a 271.9-kb region of common bean containing the SAP6 marker and found25putative genes. Functional annotation revealed 10 genes encoding proteins associated with plant defense response.However,none of these genes encoded known defense genes.Notable among them were nucleoside diphosphate kinases(housekeeping genes),phosphokinases(associated with signaling),cytochromes P450(associated with photosynthesis),RelA/SPoT(environmental stress genes),and genes encoding proteins belonging to the GRAS family and germin-like proteins(GLPs).These genes could play a role in defense via complementation with other genes.This mechanism might explain why SAP6-linked resistance is effective in some bean backgrounds but not in others.

Table 3-Effect of SU91-and BC420-linked QTL on 100-seed weight of beans in two disease environments.(Extracted from Miklas et al.[3].)

Recently,Perry et al.[36],using whole-genome sequencing of the resistant genotype OAC-Rex,identified18genes surrounding SU91 QTL that were not present in a previously sequenced susceptible genotype,G19833.No function could be assigned to seven genes,whereas 11 showed similarity with known genes and might provide selective advantage.It is puzzling to find unique genes interspersed throughout the sequences conserved between OAC-Rex and G19833 without clear hallmarks of interspecific hybridization.The unique genes with putative homology included four resistance genes,two Neimann Pick-like genes,one gene encoding chalcone reductase,one pentatricopeptide,and 1 RIN U-box domain controlled protein.The Niemann Pick-like gene is a cholesterol transporter in humans.Unlike in G19833 where the Neimann Pick-like gene is intact and has＞12 transmembrane domains,in OAC-Rex the same gene is interrupted by a 3000 bp fragment,resulting in two genes one containing five transmembrane domains and another eight(Fig.1).The gene may be functional in susceptible genotypes such as G19833 but disrupted in function in resistant genotypes such as OAC-Rex.This disruption may be a mechanism for increasing resistance by interfering with transport of materials required for pathogen growth.It has been reported by Wang et al.[20]that bacteria increase nutrient efflux into the apoplast using their effectors to enhance their pathogenicity.The Neimann Picklike gene may be one of the effector targets for manipulating host resistance.Whether this gene has a mutation in all known resistant genotypes remains to be determined.The SU91 marker from the wild bean P.acutifolius is present in several CBB-resistant genotypes sequenced to date[39]which will be helpful in future studies investigating the Neimann Pick-like gene.

Fig.1-Comparison of the regions surrounding the SU91 marker in OAC-Rex contig 232,701 with the corresponding sequence from G19833 Chr.08.Unique genes are marked in yellow,and two genes(232701-8-007 and 232701-8-008)that show sequence similarity to the G19833 Niemann Pick transporter gene are highlighted for comparison.232701-8-007 is a putative homolog of two P.acutifolius ESTs(HO787932 and HO791620),while 232701-8-008 is a putative homolog of a single P.acutifolius EST HO801643).Highly conserved genes bordering this region in G19833 and OAC Rex are labeled.The locations of molecular markers are indicated with triangles above and below the sequence.(This figure was adapted from Perry et al.[36]under a Creative Commons Attribution 4.0 International License.)

6.Expression of genes associated with CBB resistance

Studies of the expression of genes in resistance QTL during infection and disease development are limited.Cooper[37]examined genes associated with QTL linked to Pv-cttt001,but results were not conclusive.Shi et al.[34]also identified differentially expressed genes(DEGs)in leaves of the bean genotype HR45 following inoculation with CBB.DEGs comprised only 10%of transcript-derived fragments.However,50.6%of DEGs did not match any ESTs in the NCBI database.The DEGs matching ESTs in the database were associated with defense response,metabolism,photosynthesis,cellular transport,and transcription regulation.Only six DEGs could be mapped to QTL linked to BC420 which shows limited association of DEGs and this resistance marker.The few genes matching the database may be as a result of resistance genes in P.vulgaris that have been introgressed from unsequenced wild relatives.Further functional annotation and gene expression studies are needed using the available bean reference genome[40]to identify and characterize genes for CBB resistance.Transcriptome analysis using new approaches such as RNA sequencing will accelerate this process[40].There is also a need to sequence the wild bean relatives from which major resistance genes have been obtained.

7.Conclusions

The diversity of common bacterial blight disease-causing bacteria has not been exhaustively studied especially in areas where detailed molecular diversity has not been done.Molecular diversity studies could reveal further hidden secrets of these pathogens and could lead to identification of new pathogen species.Functional diversity studies of CBB-causing agents performed to date show that some isolates have coevolved with wild and others with domesticated beans.CBB isolates with capacity to overcome all known major sources of resistance to CBB are known,but their frequency in different regions of the world is unknown.It is thus important that future functional studies using bean genotypes carrying major resistance QTL be performed to guide the deployment of resistant varieties.The identified genes in the major QTLs linked to SAP6 and SU91 do not play a direct role in plant defense,given that some lack functional motifs found in conventional plant defense genes and others are conserved in both resistant and susceptible genotypes.Disrupting transport of materials required for pathogen growth may be a mechanism of resistance in genotypes carrying SU91-linked-QTL.Gene disruption as a mechanism of resistance associated with SU91-linked QTL merits further study.

Acknowledgments

The authors are grateful for research funds from the Higher Education,Science and Technology(HEST)Project of CIAT(Uganda).