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====== AvrBs2 ======

Author: [[https://www.researchgate.net/profile/Spela_Alic|Špela Alič]]\\
Internal reviewer: [[https://www.researchgate.net/profile/Ralf_Koebnik|Ralf Koebnik]]\\
Expert reviewer: FIXME

Class: AvrBs2\\
Protein family: AvrBs2\\
Prototype: AvrBs2 (//Xanthomonas euvesicatoria// pv. //euvesicatoria//, ex //Xanthomonas campestris// pv. //vesicatoria//; strain 85-10)\\
RefSeq ID: [[https://www.ncbi.nlm.nih.gov/protein/WP_011345810.1|WP_011345810.1]] (714 aa)\\
Synonym: AvrRxc1/3 (Ignatov //et al.//, 2002)\\
3D structure: Unknown

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

=== How discovered? ===

Indirectly – the pathovars that induced //Bs2//-mediated hypersensitivity were classified as having AvrBs2 activity (Kearney & Staskawicz, 1990).
=== (Experimental) evidence for being a T3E ===

Mary Beth Mudgett and coworkers provided the first evidence that AvrBs2 is secreted from //Xanthomonas campestis// pv. //vesicatoria// (//Xcv//) and that secretion is type III (//hrp//) dependent (Mudgett //et al.//, 2000). N- and C-terminal deletion analyses of AvrBs2 identified the effector domain of AvrBs2 that is recognized by //Bs2// pepper plants. By using a truncated //Pseudomonas syringae// AvrRpt2 effector reporter devoid of type III signal sequences, they localized the minimal region of AvrBs2 that is required for type III secretion in //Xcv.// Furthermore, they identified the region of AvrBs2 that is required for both type III secretion and translocation to host plants (Mudgett //et al.//, 2000). The mapping of AvrBs2 sequences sufficient for type III delivery also revealed the presence of a potential mRNA secretion signal (Mudgett //et al.//, 2000), a hypothesis that was first put forward by the lab of Olaf Schneewind (Anderson & Schneewind, 1997) and that provoked controversies over the years to come (Ghosh, 2004; Habyarimana & Ahmer, 2013).

Type III-dependent translocation of AvrBs2 was later confirmed using the calmodulin-dependent adenylate cyclase domain (Cya) of the //Bordetella pertussis// cyclolysin as a reporter (Casper-Lindley //et al//., 2002). Effector translocation into plant cells (cytosol) was detected through rise of cAMP levels inside the plant tissue of pepper plants. Mutants and //hrcV// and //hrpF// were used as a negative controls to prove that the secretion and translocation process depends on the type III secretion system (Casper-Lindley //et al//., 2002). Further it was shown that AvrBs2 contains two N-terminal secretion and translocation signals: the first for secretion and the second for enhancing translocation (Casper-Lindley //et al//., 2002).

Once the effector domain of AvrBs2 that is recognized by //Bs2// pepper plants was identified (Mudgett //et al.//, 2000), this knowledge was used to construct a Tn//5//-based reporter transposon, which was sucessfully used in genetic screens to isolate type III effectors from //Xanthomonas// (Roden //et al.//, 2004).
=== Regulation ===

Transcriptome analysis (RNA-seq) and qRT-PCR have shown that //avrBs2// gene expression is downregulated in a //X. citri// pv. //citri// Δ//phoP// mutant, indicating that PhoP is a positive regulator of //avrBs2// expression (Wei //et al//., 2019).

qRT-PCR revealed that transcript levels of 15 out of 18 tested non-TAL effector genes (as well as the regulatory genes //hrpG// and //hrpX//), including //avrBs2//, were significantly reduced in the //Xanthomonas oryzae// pv. //oryzae// Δ//xrvC// mutant compared with those in the wild-type strain PXO99<sup>A</sup>  (Liu //et al//., 2016).
=== Phenotypes ===

  * The loss of a functional //avrBs2//  gene was found to affect the fitness of //Xcv//  and revealed fitness costs for three additional, plasmid-borne effector genes (//avrBs1//, //avrBs3//, //avrBs4//) in //Xcv//, indicating that complex functional interactions exist among effector genes (Wichmann & Bergelson, 2004).
  * AvrBs2 has been demonstrated to be required for full virulence of //Xcv//, //X. oryzae//  pv. //oryzicola//, //X. phaseoli //pv. //manihotis//  (aka //X. axonopodis//  pv. //manihotis//) (Zhao //et al//., 2011; Li //et al//., 2015; Mutka //et al.//, 2016; Medina //et al//., 2018).
  * Recognition of //AvrBs2//  by OsHRL makes rice more resistant against //X. oryzae//  pv. //oryzicola//  (Park //et al//., 2010).
  * It was shown in pepper and tomato lines without //Bs2 //that mutations of catalytic residues in the glycerolphosphodiesterase did not interfere with the ability of the plant to recognize AvrBs2 through the cognate R gene //Bs2//  and trigger disease resistance. This finding suggests that recognition of AvrBs2 is independent of its glycerolphosphodiesterase enzyme activity (Zhao //et al//., 2011).
  * AvrBs2 contributes to //X. oryzae//  pv. //oryzicola//  virulence by suppressing PAMP-triggered defense responses in rice (Li //et al//., 2015).
  * AvrBs2 transiently expressed in //Arabidopsis//  protoplasts suppressed flg22-induced NHO1 expression (Li //et al//., 2015).
  * Induced expression of AvrBs2 in transgenic cell cultures was shown to dramatically suppress flg22-induced and chitin-induced immune responses, such as ROS burst and PR gene expression (Li //et al//., 2015).
  * A ∆//xopK//  mutant strain of //Xanthomonas phaseoli//  pv. //manihotis//  showed reduced growth in planta and delayed spread through the vasculature system of cassava. Moreover, the ∆//avrBs2//  mutant strain exhibited reduced water-soaking symptoms at the site of inoculation (Mutka //et al.//, 2016).

=== Localization ===

The //avrBs2//  gene is chromosomal (Coplin, 1989). The AvrBs2 protein is translocated from bacterial cells into the plant cytosol. Subcellular localization of AvrBs2 was demonstrated using recombinant AvrBs2::GFP reporter fusions transiently expressed in rice protoplasts. Green fluorescence of AvrBs2::GFP was detected across the entire cell. Similar subcellular localization was observed in //Nicotiana benthamiana//  (Li //et al//., 2015).

=== Enzymatic function ===

Shown activity of glycerolphosphodiesterase catalytic site //in-vitro//; agrocinopine synthase activity predicted but has not been experimentally confirmed (Zhao //et al//., 2011).

=== Interaction partners ===

Gene-for-gene relationship with corresponding resistance gene //Bs2//  (Minsavage //et al//., 1990). Furthermore, interaction between AvrBs2 and OsHRL was experimentaly shown by yeast two-hybrid screening (Park //et al//., 2010).

===== Conservation =====

=== In xanthomonads ===

Yes (//e.g.//, //X//. //arboricola//, //X//. //campestris//, //X//. //citri//, //X. euvesicatoria//, //X//. //fuscans//, //X. oryzae//, //X//. //phaseoli//).

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

No report.

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

=== How discovered? ===

Indirectly – the pathovars that induced //Bs2//-mediated hypersensitivity were classified as having AvrBs2 activity (Kearney & Staskawicz, 1990).
=== (Experimental) evidence for being a T3E ===

AvrBs2 fused to the calmodulin-activated adenylate cyclase domain was shown to translocate into plant cells (cytosol), detected through rise of cAMP levels inside the plant tissue. The //hrpF// <sup>-</sup>  mutant was used as a negative control to prove the translocation process. Further it was shown that AvrBs2 contains two N-terminal secretion and translocation signals: first for secretion and the second for enhancing translocation (Casper-Lindley //et al//., 2002).
=== Regulation ===

qRT-PCR revealed that transcript levels of 15 out of 18 tested non-TAL effector genes (as well as the regulatory genes //hrpG// and //hrpX//), including //avrBs2//, were significantly reduced in the //Xanthomonas oryzae// pv. //oryzae// Δ//xrvC// mutant compared with those in the wild-type strain PXO99<sup>A</sup>  (Liu //et al//., 2016).

Transcriptome analysis (RNA-seq) and qRT-PCR have shown that //avrBs2// gene expression is downregulated in a //X. citri// pv. //citri// Δ//phoP// mutant, indicating that PhoP is a positive regulator of //avrBs2// expression (Wei //et al//., 2019).
=== Phenotypes ===

  * AvrBs2 has been demonstrated to be required for full virulence of //X. euvesicatoria//  pv. //euvesicatoria//  (aka //X. campestris//  pv. //vesicatoria//), //X. oryzae//  pv. //oryzicola//, //X. phaseoli //pv. //manihotis//  (aka //X. axonopodis//  pv. //manihotis//) (Zhao //et al//., 2011; Li //et al//., 2015; Mutka //et al.//, 2016; Medina //et al//., 2018).
  * Recognition of AvrBs2 by OsHRL makes rice more resistant against //X. oryzae//  pv. //oryzicola//  (Park //et al//., 2010).
  * It was shown in pepper and tomato lines without //Bs2 //that mutations of catalytic residues in the glycerolphosphodiesterase did not interfere with the ability of the plant to recognize AvrBs2 through the cognate R gene //Bs2//  and trigger disease resistance. This finding suggests that recognition of AvrBs2 is independent of its glycerolphosphodiesterase enzyme activity (Zhao //et al//., 2011).
  * AvrBs2 contributes to //X. oryzae//  pv. //oryzicola//  virulence by suppressing PAMP-triggered defense responses in rice (Li //et al//., 2015).
  * AvrBs2 transiently expressed in //Arabidopsis//  protoplasts suppressed flg22-induced NHO1 expression (Li //et al//., 2015).
  * Induced expression of AvrBs2 in transgenic cell cultures was shown to dramatically suppress flg22-induced and chitin-induced immune responses, such as ROS burst and PR gene expression (Li //et al//., 2015).
  * A ∆//xopK//  mutant strain of //Xanthomonas phaseoli//  pv. //manihotis//  (aka //Xanthomonas axonopodis//  pv. //manihotis//) showed reduced growth in planta and delayed spread through the vasculature system of cassava. Moreover, the ∆avrBs2 mutant strain exhibited reduced water-soaking symptoms at the site of inoculation (Mutka //et al.//, 2016).
  * //Agrobacterium//-mediated transient expression of both XopQ and XopX in rice cells resulted in induction of rice immune responses, which were not observed when either protein was individually expressed. A screen for //Xanthomonas//  effectors which can suppress XopQ-XopX induced rice immune responses, led to the identification of five effectors, namely XopU, XopV, XopP, XopG and AvrBs2, that could individually suppress these immune responses. These results suggest a complex interplay of //Xanthomonas//  T3SS effectors in suppression of both pathogen-triggered immunity and effector-triggered immunity to promote virulence on rice (Deb //et al.//, 2020).
=== Localization ===

The //avrBs2//  gene is chromosomal (Coplin, 1989). The AvrBs2 protein is translocated from bacterial cells into the plant cytosol. Subcellular localization of AvrBs2 was demonstrated using recombinant AvrBs2::GFP reporter fusions transiently expressed in rice protoplasts. Green fluorescence of AvrBs2::GFP was detected across the entire cell. Similar subcellular localization was observed in //Nicotiana benthamiana//  (Li //et al//., 2015).

=== Enzymatic function ===

Shown activity of glycerolphosphodiesterase catalytic site //in-vitro//; agrocinopine synthase activity predicted but has not been experimentally confirmed (Zhao //et al//., 2011).

=== Interaction partners ===

Gene-for-gene relationship with corresponding resistance gene //Bs2//  (Minsavage //et al//., 1990). Furthermore, interaction between AvrBs2 and OsHRL was experimentaly shown by yeast two-hybrid screening (Park //et al//., 2010).

===== Conservation =====

=== In xanthomonads ===

Yes (//e.g.//, //X//. //arboricola//, //X//. //campestris//, //X//. //citri//, //X. euvesicatoria//, //X//. //fuscans//, //X. oryzae//, //X//. //phaseoli//).

Field strains of //X. euvesicatoria// pv. //euvesicatoria// and //X//. //campestris// pv. //campestris// were found to accumulate mutations in the //avrBs2/////avrRxc1/3// gene in order to overcome //Bs2/////Rxc1/////Rxc3//-mediated resistance (Swords //et al.//, 1996; Gassmann //et al.//, 2000; Ignatov //et al.//, 2002). Yet, the global //Xcv// population was found to be extremely clonal, with very little genetic variation throughout the chromosome, including //avrBs2// and the plasmid-borne //avrBs1//, a finding that is consistent with recent evolution or population expansion of the species (Wichmann //et al.//, 2005).
=== In other plant pathogens/symbionts ===

No report.

===== References =====

Anderson DM, Schneewind O (1997). A mRNA signal for the type III secretion of Yop proteins by //Yersinia enterocolitica//. Science 278: 1140-1143. DOI: [[https://doi.org/10.1126/science.278.5340.1140|10.1126/science.278.5340.1140]]

Casper-Lindley C. Dahlbeck D, Clark ET, Staskawicz BJ (2002). Direct biochemical evidence for type III secretion-dependent translocation of the AvrBs2 effector protein into plant cells. Proc. Natl. Acad. Sci. USA 99: 8336-8341. DOI: [[https://doi.org/10.1073/pnas.122220299|10.1073/pnas.122220299]]

Coplin DL (1989). Plasmids and their role in the evolution of plant pathogenic bacteria. Ann. Rev. Phytopathol. 27: 187-212. DOI: [[https://doi.org/10.1146/annurev.py.27.090189.001155|10.1146/annurev.py.27.090189.001155]]

Deb S, Ghosh P, Patel HK, Sonti RV (2020). Interaction of the //Xanthomonas// effectors XopQ and XopX results in induction of rice immune responses. Plant J., in press. DOI: [[https://doi.org/10.1111/tpj.14924|10.1111/tpj.14924]]

Gassmann W, Dahlbeck D, Chesnokova O, Minsavage GV, Jones JB, Staskawicz BJ (2000). Molecular evolution of virulence in natural field strains of //Xanthomonas campestris// pv. //vesicatoria//. J. Bacteriol. 182: 7053-7059. DOI: [[https://doi.org/10.1128/jb.182.24.7053-7059.2000|10.1128/jb.182.24.7053-7059.2000]]

Ghosh P (2004). Process of protein transport by the type III secretion system. Microbiol. Mol. Biol. Rev. 68: 771-795. DOI: [[https://doi.org/10.1128/MMBR.68.4.771-795.2004|10.1128/MMBR.68.4.771-795.2004]]

Habyarimana F, Ahmer BM (2013). More evidence for secretion signals within the mRNA of type 3 secreted effectors. J. Bacteriol. 195: 2117-2118. DOI: [[https://doi.org/10.1128/JB.00303-13|10.1128/JB.00303-13]]

Ignatov AN, Monakhos GF, Dzhalilov FS, Pozmogova GV (2002). Avirulence gene from //Xanthomonas campestris //pv. //campestris// homologous to the //avrBs2// locus is recognized in race-specific reaction by two different resistance genes in Brassicas. Genetika 38: 1656-1662 [Article in Russian] / Russian J. Genet. 38: 1404-1410. DOI: [[https://doi.org/10.1023/A:1021643907032|10.1023/A:1021643907032]]

Kearney B, Staskawicz BJ (1990). Widespread distribution and fitness contribution of //Xanthomonas campestris// avirulence gene //avrBs2//. Nature 346: 385-386. DOI: [[https://doi.org/10.1038/346385a0|10.1038/346385a0]]

Li S, Wang Y, Wang S, Fang A, Wang J, Liu L, Zhang K, Mao Y, Sun W (2015). The type III effector AvrBs2 in //Xanthomonas oryzae// pv. //oryzicola// suppresses rice immunity and promotes disease development. Mol. Plant Microbe Interact. 28: 869-880. DOI: [[https://doi.org/10.1094/MPMI-10-14-0314-R|10.1094/MPMI-10-14-0314-R]]

Liu Y, Long J, Shen D, Song C (2016). //Xanthomonas oryzae// pv. //oryzae// requires H-NS-family protein XrvC to regulate virulence during rice infection. FEMS Microbiol. Lett. 363: fnw067. DOI: [[https://doi.org/10.1093/femsle/fnw067|10.1093/femsle/fnw067]]

Medina CA, Reyes PA, Trujillo CA, Gonzalez JL, Bejarano DA, Montenegro NA, Jacobs JM, Joe A, Restrepo S, Alfano JR, Bernal A (2018). The role of type III effectors from //Xanthomonas axonopodis// pv. //manihotis// in virulence and suppression of plant immunity. Mol. Plant Pathol. 19: 593-606. DOI: [[https://doi.org/10.1111/mpp.12545|10.1111/mpp.12545]]

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: [[https://doi.org/10.1094/MPMI-3-041|10.1094/MPMI-3-041]]

Mudgett MB, Chesnokova O, Dahlbeck D, Clark ET, Rossier O, Bonas U, Staskawicz BJ (2000). Molecular signals required for type III secretion and translocation of the //Xanthomonas campestris// AvrBs2 protein to pepper plants. Proc. Natl. Acad. Sci. USA 97: 13324-13329. DOI: [[https://doi.org/10.1073/pnas.230450797|10.1073/pnas.230450797]]

Mutka AM, Fentress SJ, Sher JW, Berry JC, Pretz C, Nusinow DA, Bart R (2016). Quantitative, image-based phenotyping methods provide insight into spatial and temporal dimensions of plant disease. Plant Physiol. 172: 650-660. DOI: [[https://doi.org/10.1104/pp.16.00984|10.1104/pp.16.00984]]

Park SR, Moon SJ, Shin DJ, Kim MG, Hwang DJ, Bae SC, Kim JG , Yi BY, Byun MO (2010). Isolation and characterization of rice //OsHRL// gene related to bacterial blight resistance. Plant Pathol. J. 26: 417-420. DOI: [[https://doi.org/10.5423/PPJ.2010.26.4.417|10.5423/PPJ.2010.26.4.417]]

Roden JA, Belt B, Ross JB, Tachibana T, Vargas J, Mudgett MB (2004). A genetic screen to isolate type III effectors translocated into pepper cells during //Xanthomonas// infection. Proc. Natl. Acad. Sci. USA 101: 16624-16629. DOI: [[https://doi.org/10.1073/pnas.0407383101|10.1073/pnas.0407383101]]

Swords KM, Dahlbeck D, Kearney B, Roy M, Staskawicz BJ (1996). Spontaneous and induced mutations in a single open reading frame alter both virulence and avirulence in //Xanthomonas campestris// pv. //vesicatoria// //avrBs2//. J. Bacteriol. 178: 4661-4669. DOI: [[https://doi.org/10.1128/jb.178.15.4661-4669.1996|10.1128/jb.178.15.4661-4669.1996]]

Wei C, Ding T, Chang C, Yu C, Li X, Liu Q (2019). Global regulator PhoP is necessary for motility, biofilm formation, exoenzyme production and virulence of //Xanthomonas citri// subsp. //citri// on citrus plants. Genes 10: 340. DOI: [[https://doi.org/10.3390/genes10050340|10.3390/genes10050340]]

Wichmann G, Bergelson J (2004). Effector genes of //Xanthomonas axonopodis// pv. //vesicatoria// promote transmission and enhance other fitness traits in the field. Genetics 166: 693-706. DOI: [[https://doi.org/10.1534/genetics.166.2.693|10.1534/genetics.166.2.693]]

Wichmann G, Ritchie D, Kousik CS, Bergelson J (2005). Reduced genetic variation occurs among genes of the highly clonal plant pathogen //Xanthomonas axonopodis// pv. //vesicatoria//, including the effector gene //avrBs2//. Appl. Environ. Microbiol. 71: 2418-2432. DOI: [[https://doi.org/10.1128/AEM.71.5.2418-2432.2005|10.1128/AEM.71.5.2418-2432.2005]]

Zhao B, Dahlbeck D, Krasileva KV, Fong RW, Staskawicz BJ (2011). Computational and biochemical analysis of the //Xanthomonas// effector AvrBs2 and its role in the modulation of //Xanthomonas// type three effector delivery. PLoS Pathog. 7: e1002408. DOI: [[https://doi.org/10.1371/journal.ppat.1002408|10.1371/journal.ppat.1002408]]

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

Timilsina S, Abrahamian P, Potnis N, Minsavage GV, White FF, Staskawicz BJ, Jones JB, Vallad GE, Goss EM (2016). Analysis of sequenced genomes of //Xanthomonas perforans//  identifies candidate targets for resistance breeding in tomato. Phytopathology 106: 1097-1104. DOI: [[https://doi.org/10.1094/PHYTO-03-16-0119-FI|10.1094/PHYTO-03-16-0119-FI]]. Corrected in: Phytopathology (2019) 109: 1820. DOI: [[https://doi.org/10.1094/PHYTO-03-16-0119.1-FI|10.1094/PHYTO-03-16-0119.1-FI]]
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