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Molecular Diagnosis and Diversity for Regulated Xanthomonas


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bacteria:t3e:avrbs2

AvrBs2

Author: Špela Alič
Internal reviewer: 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: 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 Tn5-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 ΔphoP 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 ΔxrvC mutant compared with those in the wild-type strain PXO99A (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 - 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 ΔxrvC mutant compared with those in the wild-type strain PXO99A (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 ΔphoP 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 10.1094/PHYTO-03-16-0119-FI. Corrected in: Phytopathology (2019) 109: 1820. DOI: 10.1094/PHYTO-03-16-0119.1-FI

bacteria/t3e/avrbs2.txt · Last modified: 2020/07/17 10:32 by rkoebnik