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====== AvrBs3 ======

Author: [[https://www.researchgate.net/profile/Nay_Dia2|Nay C. Dia]]\\
Internal reviewer: [[https://www.genetik.uni-hannover.de/boch.html|Jens Boch]]\\
Expert reviewer: [[https://www.researchgate.net/profile/Sabine_Thieme3|Sabine Thieme]]

Class: AvrBs3\\
Family: Transcription Activator-Like (TAL) Effectors, TALEs (previously: AvrBs3/PthA)\\
Prototype: AvrBs3 (//Xanthomonas euvesicatoria// pv. //euvesicatoria//, ex //Xanthomonas campestris// pv. //vesicatoria//; strain 71-21)\\
RefSeq ID: [[https://www.ncbi.nlm.nih.gov/protein/P14727.2|P14727.2]] (1164 aa)\\
3D structure: [[https://www.rcsb.org/structure/2KQ5|2KQ5]] (Murakami //et al.//, 2010); [[https://www.rcsb.org/structure/3V6P|3V6P]], [[https://www.rcsb.org/structure/3V6T| 3V6T ]] (Deng //et al.//, 2012a); [[https://www.rcsb.org/structure/4GJP|4GJP]], [[https://www.rcsb.org/structure/4GJR|4GJR]] (Deng //et al.//, 2012b); [[https://www.rcsb.org/structure/4HPZ|4HPZ]] (Gao //et al.//, 2012) ; [[https://www.rcsb.org/structure/3UGM|3UGM]] (Mak //et al.//, 2012); [[https://www.rcsb.org/structure/4GG4|4GG4]] (Yin //et al.//, 2012); [[https://www.rcsb.org/structure/2YPF|2YPF ]] (Stella //et al//., 2013); [[https://www.rcsb.org/structure/4OSH|4OSH]], [[https://www.rcsb.org/structure/4OSI| 4OSI]], [[https://www.rcsb.org/structure/4OSJ| 4OSJ]], [[https://www.rcsb.org/structure/4OSK| 4OSK]], [[https://www.rcsb.org/structure/4OSL| 4OSL]], [[https://www.rcsb.org/structure/4OSM| 4OSM]], [[https://www.rcsb.org/structure/4OSQ|4OSQ]], [[https://www.rcsb.org/structure/4OSR|4OSR]], [[https://www.rcsb.org/structure/4OSS|4OSS]], [[https://www.rcsb.org/structure/4OST| 4OST]], [[https://www.rcsb.org/structure/4OSV| 4OSV]], [[https://www.rcsb.org/structure/4OSW| 4OSW]], [[https://www.rcsb.org/structure/4OSZ| 4OSZ]], [[https://www.rcsb.org/structure/4OT0| 4OT0]], [[https://www.rcsb.org/structure/4OT3| 4OT3]], [[https://www.rcsb.org/structure/4OTO|4OTO]] (Deng //et al.//, 2014); [[https://www.rcsb.org/structure/6JTQ|6JTQ]], [[https://www.rcsb.org/structure/6JVZ|6JVZ]], [[https://www.rcsb.org/structure/6JW0| 6JW0]], [[https://www.rcsb.org/structure/6JW1|6JW1]], [[https://www.rcsb.org/structure/6JW2|6JW2]], [[https://www.rcsb.org/structure/6JW3|6JW3]], [[https://www.rcsb.org/structure/6JW4|6JW4]], [[https://www.rcsb.org/structure/6JW5|6JW5]] (Liu & Yi, unpublished)

===== Biological function =====

=== How discovered? ===

The gene //avrBs3 //was cloned in 1989 and was the first gene described of the TAL effector (TALE) family (Minsavage //et al//., 1990). Different resistant and susceptible cultivars of peppers were inoculated with //Xcv// strains 71-21 and 82-8 (Bonas //et al//., 1989). The pepper cultivar ECW-30R carries the resistance gene //Bs3 //and inoculation of these //Xcv// strains provoked a hypersensitive response (HR) (Bonas //et al//., 1989). This indicated that both //Xcv// strains contained //avrBs3//.
=== (Experimental) evidence for being a T3E ===

AvrBs3 is secreted and translocated into the plant via the Hrp type III secretion system (Bonas //et al//., 1991; Van den Ackerveken //et al//., 1996; Bonas //et al//., 1999). In contrast to wild-type bacteria, an //Xcv// mutant carrying a deletion in the conserved //hrp// gene //hrcV// did not secrete AvrBs3 indicating that AvrBs3 is transported by the Hrp system (Rossier //et al//., 1999). The first 10 and 50 amino acids of AvrBs3 are required for secretion and translocation, respectively (Scheibner //et al//., 2017). In its C-terminal domain, AvrBs3 carries an acidic activation domain which is functional in plant cells (Van den Ackerveken //et al//., 1996). Two nuclear localization signals in the C-terminal domain of AvrBs3 facilitate transport into the plant cell nucleus (Van den Ackerveken //et al//., 1996; Szurek //et al//., 2002). These eukaryotic features support the role of AvrBs3 and members of the TALE family within the eukaryotic host cell.
=== Regulation ===

Unlike most other type III effectors, expression of //avrBs3// is not dependend on the hrp regulon and the gene does not contain a PIP box in its promoter region. It is expressed constitutively in cells grown in minimal or complex medium and in planta (Knoop //et al//., 1991).
=== Phenotypes ===

AvrBs3, as well as other members of the TALE family, function as specific transcription factors in plant cells. These proteins bind to specific sequences in promoters and induce expression of downstream genes. The DNA-binding specificity is encoded in the order of individual 34-amino acid repeats which each recognize one DNA base. Different TALEs typically contain different repeats and accordingly bind to different DNA sequences and target different host genes. The contributions of individual TALEs to virulence can thus be quite diverse.

Expression analysis using gene promoter fusion and western blot analysis demonstrated that //avrBs3// was expressed and resulted in a 122 kDa protein (1164 aa) which was detectable using a specific polyclonal antibody (Bonas //et al//., 1989). The AvrBs3 effector protein elicits two different types of responses in resistant or susceptible plants. In susceptible pepper plants (Early Cal Wonder; ECW), hypertrophy (i.e. enlargement of mesophyll cells) is triggered by AvrBs3 (Bonas //et al//., 1989; Bonas //et al//., 1991; Marois //et al//., 2002). //Agrobacterium// strains carrying a vector with //avrBs3// induced pustules (hypertrophy) 4-5 dpi in various solanaceous plants including //Nicotiana// //clevelandii//, //N.// //benthamiana//, //N.// //tabacum//, //Petunia hybrida//, //Physalis alkekengi//, //Solanum americanum// and potato (//S.// //tuberosum//), whereas //Agrobacterium// strains carrying an empty vector did not cause any changes in inoculated plants (Marois //et al//., 2002; Kay //et al//., 2007). Differential cDNA analysis from susceptible pepper plants infected with //Xcv// with or without AvrBs3 led to the discovery of //upa// (upregulated by AvrBs3) genes whose expression is induced by AvrBs3 (Marois //et al.//, 2002; Kay //et al//., 2007). These //UPA// genes all share a conserved promoter element, known as the //UPA// box (Kay //et al.//, 2007). //UPA20// acts as a master regulator of cell enlargement causing the hypertrophy symptoms associated with AvrBs3. Silencing of //UPA20// decreased cell hypertrophy in infected plants while the expression of //UPA20// led to hypertrophy in uninfected plants (Kay //et al//., 2007).

In resistant pepper plants, the promoter of //Bs3// contains a //UPA// box that is bound by AvrBs3 resulting in the transcription of the gene //Bs3//. //Bs3// encodes a protein that is homologous to flavine-dependent mono-oxygenases (Römer //et al//., 2007) and its expression causes rapid cell death thus preventing the spread of the pathogen (Bonas //et al//., 1989; Bonas //et al//., 1991).

The central region of the //avrBs3// gene consists of 17.5 nearly identical 102 bp repeats. Each repeat encodes 34 amino acids (Bonas //et al//., 1989). Repeat variable di-residues (RVDs) at positions 12 and 13 determine the specificity of each repeat (Boch //et al//., 2009; Moscou & Bogdanove, 2009). Rearranging individual repeats enables construction of any desired DNA-binding specificity (Boch //et al.//, 2009).
=== Localization ===

The //avrBs3// gene is localized on pXV11, a self-transmissible plasmid, and was initially isolated from //Xcv// strain 71-21 (Bonas //et al//., 1989). Using complementation of //Xcv// strain 85-10 (virulent on pepper ECW-30R), a 5-kb fragment including //avrBs3// was discovered (Bonas //et al//., 1989).
=== Molecular function ===

DNA-binding protein. Transcriptional activator.

=== Interaction partners ===

Importin alpha (Szurek //et al.//, 2001) interacts with the nuclear localization sequences of AvrBs3. The basal transcription factor IIA, gamma subunit from rice interacts with a region in the C-terminal domain of TALEs (Yuan //et al//., 2016) and similar interactions might be possible for AvrBs3, too. AvrBs3 and the TALE-family of effectors bind to DNA (Kay //et al//., 2007; Römer //et al//., 2007) with their N-terminal domain exhibiting general DNA-binding properties (Gao //et al.//, 2012) and the repeat region facilitating specific interaction to DNA bases (Boch //et al//., 2009; Moscou & Bogdanove, 2009).
===== Conservation =====

=== In xanthomonads ===

Yes, in many pathovars, but not necesssarily all strains within a pathovar.

=== In other plant pathogens/symbionts ===

Yes: Genes homologous to //avrBs3// of //Xanthomonas// were detected in some strains of //Ralstonia solanacearum// biovars 3, 4 and 5 (Heuer //et al//., 2007), in endofungal strains of //Burkholderia rhizoxinica // (Lacker //et al//., 2011), and in unknown marine organisms. All these related proteins can bind DNA (de Lange //et al//., 2013; de Lange //et al.//, 2014; de Lange //et al//., 2015).
===== References =====

Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U (2009). Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326: 1509-1512. DOI: [[http://dx.doi.org/10.1126/science.1178811|10.1126/science.1178811]]

Bonas U, Stall, RE, Staskawicz B (1989). Genetic and structural characterization of the avirulence gene //avrBs3// from //Xanthomonas campestris// pv. //vesicatoria//. Mol. Gen. Genet. 218: 127-136. DOI: [[http://dx.doi.org/10.1007/BF00330575|10.1007/BF00330575]]

Bonas U, Schulte R, Fenselau S, Minsavage GV, Staskawicz BJ, Stall RE (1991). Isolation of a gene cluster from //Xanthomonas// //campestris// pv. //vesicatoria// that determines pathogenicity and the hypersensitive response on pepper and tomato. Mol. Plant Microbe Interact 4: 81-88. DOI: [[http://dx.doi.org/10.1094/MPMI-4-081|10.1094/MPMI-4-081]]

Bonas U, Van den Ackerveken G (1999). Gene-for-gene interactions: bacterial avirulence proteins specify plant disease resistance. Curr. Opin. Microbiol. 2: 94-98. DOI: [[https://doi.org/10.1016/S1369-5274(99)80016-2|10.1016/S1369-5274(99)80016-2]]

de Lange O, Schreiber T, Schandry N, Radeck J, Braun KH, Koszinowski J, Heuer H, Strauß A, Lahaye T (2013). Breaking the DNA-binding code of //Ralstonia solanacearum// TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease. New Phytol. 199: 773-786. DOI: [[https://doi.org/10.1111/nph.12324|10.1111/nph.12324]]

de Lange O, Wolf C, Dietze J, Elsaesser J, Morbitzer R, Lahaye T (2014). Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain. Nuc. Acids Res. 42: 7436-7449. DOI: [[https://doi.org/10.1093/nar/gku329.|10.1093/nar/gku329.]]

de Lange O, Wolf C, Thiel P, Krüger J, Kleusch C, Kohlbacher O, Lahaye T (2015). DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats. Nuc. Acids Res. 43: 10065-10080. DOI: [[https://doi.org/10.1093/nar/gkv1053|10.1093/nar/gkv1053]]

Deng D, Yan C, Pan X, Mahfouz M, Wang J, Zhu JK, Shi Y, Yan N (2012a). Structural basis for sequence-specific recognition of DNA by TAL effectors. Science 335: 720-723. DOI: [[https://doi.org/10.1126/science.1215670|10.1126/science.1215670]]

Deng D, Yan C, Wu J, Pan X, Yan N (2014). Revisiting the TALE repeat. Protein Cell 5: 297-306. DOI: [[https://doi.org/10.1007/s13238-014-0035-2|10.1007/s13238-014-0035-2]]

Deng D, Yin P, Yan C, Pan X, Gong X, Qi S, Xie T, Mahfouz M, Zhu JK, Yan N, Shi Y (2012b). Recognition of methylated DNA by TAL effectors. Cell Res. 22: 1502-1504. DOI: [[https://doi.org/10.1038/cr.2012.127|10.1038/cr.2012.127]]

Gao H, Wu X, Chai J, Han Z (2012). Crystal structure of a TALE protein reveals an extended N-terminal DNA binding region. Cell Res. 22: 1716-1720. DOI: [[https://doi.org/10.1038/cr.2012.156|10.1038/cr.2012.156]]

Herbers K, Conrads-Strauch J, Bonas U (1992). Race-specificity of plant resistance to bacterial spot disease determined by repetitive motifs in a bacterial avirulence protein. Nature 356: 172-174. DOI: [[https://doi.org/10.1038/356172a0|10.1038/356172a0]]

Heuer H, Yin YN, Xue QY, Smalla K, Guo JH (2007). Repeat domain diversity of avrBs3-like genes in //Ralstonia// //solanacearum// strains and association with host preferences in the field. Appl. Environ. Microbiol. 73: 4379-4384. DOI: [[http://dx.doi.org/10.1128/AEM.00367-07|10.1128/AEM.00367-07]]

Hopkins CM, White FF, Choi SH, Guo A, Leach JE (1992). Identification of a family of avirulence genes from //Xanthomonas// //oryzae// pv. //oryzae//. Mol. Plant Microbe Interact. 5: 451-459. DOI: [[https://doi.org/10.1094/mpmi-5-451|10.1094/mpmi-5-451]]

Kay S, Hahn S, Marois E, Hause G, Bonas U (2007). A bacterial effector acts as a plant transcription factor and induces a cell size regulator. Science 318: 648-651. DOI: [[http://dx.doi.org/10.1126/science.1144956|10.1126/science.1144956]]

Knoop V, Staskawicz B, Bonas U (1991). Expression of the avirulence gene //avrBs3// from //Xanthomonas// //campestris// pv. //vesicatoria// is not under the control of the //hrp// genes and is independent of plant factors. J. Bacteriol. 173: 7142-7150. DOI: [[http://dx.doi.org/10.1128/jb.173.22.7142-7150.1991|10.1128/jb.173.22.7142-7150.1991]]

Lackner G, Moebius N, Partida-Martinez LP, Boland S, Hertweck C (2011). Evolution of an endofungal lifestyle: Deductions from the //Burkholderia rhizoxinica// genome. BMC Genomics 12: 210. DOI: [[https://doi.org/10.1186/1471-2164-12-210|10.1186/1471-2164-12-210]]

Mak AN, Bradley P, Cernadas RA, Bogdanove AJ, Stoddard BL (2012). The crystal structure of TAL effector PthXo1 bound to its DNA target. Science 335: 716-719. DOI: [[https://doi.org/10.1126/science.1216211|10.1126/science.1216211]]

Marois E, Van den Ackerveken G, Bonas U (2002). The //Xanthomonas// type III effector protein AvrBs3 modulates plant gene expression and induces cell hypertrophy in the susceptible host. Mol. Plant Microbe Interact. 15: 637-646. DOI: [[https://doi.org/10.1094/MPMI.2002.15.7.637|10.1094/MPMI.2002.15.7.637]]

Minsavage GV, Dahlbeck D, Whalen MC, Kearney B, Bonas U, Staskawicz BJ, Stall, RE (1990). Gene-for-gene relationships specifying disease resistance in //Xanthomonas// //campestris// pv. //vesicatoria//-pepper interactions. Mol. Plant Microbe Interact. 3: 41-47. DOI: [[http://dx.doi.org/10.1094/MPMI-3-041|10.1094/MPMI-3-041]]

Moscou MJ, Bogdanove AJ (2009). A simple cipher governs DNA recognition by TAL effectors. Science 326: 1501. DOI: [[http://dx.doi.org/10.1126/science.1178817|10.1126/science.1178817]]

Murakami MT, Sforça ML, Neves JL, Paiva JH, Domingues MN, Pereira AL, Zeri AC, Benedetti CE (2010). The repeat domain of the type III effector protein PthA shows a TPR-like structure and undergoes conformational changes upon DNA interaction. Proteins 78: 3386-3395. DOI: [[https://doi.org/10.1002/prot.22846|10.1002/prot.22846]]

Römer P, Hahn S, Jordan T, Strauss T, Bonas U, Lahaye T (2007). Plant pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene. Science 318: 645-648. DOI: [[https://doi.org/10.1126/science.1144958|10.1126/science.1144958]]

Rossier O, Wengelnik K, Hahn K, Bonas U (1999). The //Xanthomonas// Hrp type III system secretes proteins from plant and mammalian bacterial pathogens. Proc. Natl. Acad. Sci. USA 96: 9368-9373. DOI: [[https://doi.org/10.1073/pnas.96.16.9368|10.1073/pnas.96.16.9368]]

Scheibner F, Marillonnet S, Büttner D (2017). The TAL effector AvrBs3 from //Xanthomonas campestris// pv. //vesicatoria// contains multiple export signals and can enter plant cells in the absence of the type III secretion translocon. Front Microbiol. 8: 2180. DOI: [[https://doi.org/10.3389/fmicb.2017.02180|10.3389/fmicb.2017.02180]]

Stella S, Molina R, Yefimenko I, Prieto J, Silva G, Bertonati C, Juillerat A, Duchateau P, Montoya G (2013). Structure of the AvrBs3–DNA complex provides new insights into the initial thymine-recognition mechanism. Acta Cryst. 69: 1707-1716. DOI: [[http://dx.doi.org/10.1107/S0907444913016429|10.1107/S0907444913016429]]

Szurek B, Marois E, Bonas U, Van den Ackerveken G (2001). Eukaryotic features of the //Xanthomonas// type III effector AvrBs3: protein domains involved in transcriptional activation and the interaction with nuclear import receptors from pepper. Plant J. 26: 523-534. DOI: [[https://10.1046/j.0960-7412.2001.01046.x|10.1046/j.0960-7412.2001.01046.x]]

Szurek B, Rossier O, Hause G, Bonas U (2002). Type III-dependent translocation of the //Xanthomonas// AvrBs3 protein into the plant cell. Mol. Microbiol. 46: 13-23. DOI: [[https://doi.org/10.1046/j.1365-2958.2002.03139.x|10.1046/j.1365-2958.2002.03139.x]]

Van den Ackerveken G, Marois E, Bonas U (1996). Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell. Cell 87: 1307-1316. DOI: [[https://doi.org/10.1016/S0092-8674(00)81825-5|10.1016/S0092-8674(00)81825-5]]

Yin P, Deng D, Yan C, Pan X, Xi JJ, Yan N, Shi Y (2012). Specific DNA-RNA hybrid recognition by TAL effectors. Cell Rep. 2: 707-713. DOI: 1[[https://doi.org/0.1016/j.celrep.2012.09.001|0.1016/j.celrep.2012.09.001]]

===== Further reading =====

Boch J, Bonas U (2010). //Xanthomonas// AvrBs3 family-type III effectors: discovery and function. Annu. Rev Phytopathol. 48: 419-436. DOI: [[https://doi.org/10.1146/annurev-phyto-080508-081936|10.1146/annurev-phyto-080508-081936]]

Boch J, Bonas U, Lahaye T (2014). TAL effectors - pathogen strategies and plant resistance engineering. New Phytol. 204: 823-832. DOI: [[https://doi.org/10.1111/nph.13015|10.1111/nph.13015]]

Bogdanove AJ, Schornack S, Lahaye T (2010). TAL effectors: finding plant genes for disease and defense. Curr. Opin. Plant Biol. 13: 394-401. DOI: [[https://doi.org/10.1016/j.pbi.2010.04.010|10.1016/j.pbi.2010.04.010]]

Doyle EL, Stoddard BL, Voytas DF, Bogdanove AJ (2013). TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA-targeting proteins. Trends Cell Biol. 23: 390-398. DOI: [[https://doi.org/10.1016/j.tcb.2013.04.003|10.1016/j.tcb.2013.04.003]]

Hutin M, Pérez-Quintero AL, Lopez C, Szurek B (2015). MorTAL Kombat: the story of defense against TAL effectors through loss-of-susceptibility. Front. Plant Sci. 6: 535. DOI: [[https://doi.org/10.3389/fpls.2015.00535|10.3389/fpls.2015.00535]]. Erratum in: Front Plant Sci. (2015) 6: 647.

Xue J, Lu Z, Liu W, Wang S, Lu D, Wang X, He X (2020). The genetic arms race between plant and //Xanthomonas//: lessons learned from TALE biology. Sci. China Life Sci. 63. DOI: [[https://doi.org/10.1007/s11427-020-1699-4|10.1007/s11427-020-1699-4]]

Zhang J, Yin Z, White F (2015). TAL effectors and the executor //R// genes. Front. Plant Sci. 6: 641. DOI: [[https://doi.org/10.3389/fpls.2015.00641|10.3389/fpls.2015.00641]]
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