The Type III Effector AvrBs3 from //Xanthomonas//

The Type III Effector AvrBs3 from //Xanthomonas//

Author: Nay C. Dia
Internal reviewer: Jens Boch
Expert reviewer: 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)
GenBank ID: P14727.2 (1164 aa)
RefSeq ID: WP_011052943.1 (1126 aa)
3D structure: 2KQ5 (Murakami et al., 2010); 3V6P, 3V6T (Deng et al., 2012a); 4GJP, 4GJR (Deng et al., 2012b); 4HPZ (Gao et al., 2012); 3UGM (Mak et al., 2012); 4GG4 (Yin et al., 2012); 2YPF (Stella et al., 2013); 4OSH, 4OSI, 4OSJ, 4OSK, 4OSL, 4OSM, 4OSQ, 4OSR, 4OSS, 4OST, 4OSV, 4OSW, 4OSZ, 4OT0, 4OT3, 4OTO (Deng et al., 2014); 6JTQ, 6JVZ, 6JW0, 6JW1, 6JW2, 6JW3, 6JW4, 6JW5 (Liu et al., 2020); 6LEW (unpublished)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

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Acknowledgements

This fact sheet is based upon work from COST Action CA16107 EuroXanth, supported by COST (European Cooperation in Science and Technology).

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