Domain Dissection of AvrRxo1 for Suppressor, Avirulence and Cytotoxicity Functions

<div><p>AvrRxo1, a type III effector from <i>Xanthomonas oryzae</i> pv. <i>oryzicola</i> (Xoc) which causes bacterial leaf streak (BLS) in rice, can be recognised by non-host resistance protein Rxo1. It triggers a hypersensitive response (HR) in maize. Little is known regarding the virulence function of AvrRxo1. In this study, we determined that AvrRxo1 is able to suppress the HR caused by the non-host resistance recognition of <i>Xanthomonas oryzae</i> pv. <i>oryzae</i> (Xoo) by <i>Nicotiana benthamiana</i>. It is toxic, inducing cell death from transient expression in <i>N. benthamiana</i>, as well as in yeast. Among the four AvrRxo1 alleles from different Xoc strains, we concluded that the toxicity is abolished by a single amino acid substitution at residue 344 in two AvrRxo1 alleles. A series of truncations from the carboxyl terminus (C-terminus) indicate that the complete C-terminus of AvrRxo1 plays an essential role as a suppressor or cytotoxic protein. The C-terminus was also required for the avirulence function, but the last two residues were not necessary. The first 52 amino acids of N-terminus are unessential for toxicity. Point mutagenesis experiments indicate that the ATP/GTP binding site motif A is required for all three functions of AvrRxo1, and NLS is required for both the avirulence and the suppression of non-host resistance. The putative thiol protease site is only required for the cytotoxicity function. These results determine that AvrRxo1 plays a role in the complex interaction with host proteins after delivery into plant cells.</p></div

The transient expression of AvrRxo1 mutant proteins in <i>Nicotiana benthamiana</i>.

<p>The expressions of avrRxo1_RS105 (1), pGR106 (2) in <i>N. benthamiana</i> cells were used as positive and negative controls, respectively. Similarly hereinafter. (A) The C-terminus-truncated proteins AvrRxo1(1–159) (3), AvrRxo1(1–193) (4), AvrRxo1(1–278) (5), AvrRxo1(1–373) (6), AvrRxo1(1–412) (7) and AvrRxo1(1–420) (8) expressed in <i>N. benthamiana</i> all failed to induce cell death. (B) The transient expression of N-terminus truncated proteins in <i>N. benthamiana</i>. The expressions of AvrRxo1(52–421) (3) can still induce cell death, whereas AvrRxo1(109–421) (4) and AvrRxo1(140–421) (5) loss the toxic ability in <i>N. benthamiana</i>. (C) The transient expression of mutant proteins in <i>N. benthamiana</i>. Transient expression of avrRxo1(G165A) (3), avrRxo1(K166N) (4), and avrRxo1(H71A) (6) did not induce cell death, however expression of avrRxo1(nls) (5) still resulted in cell death in <i>N. benthamiana</i>. The normal expression of <i>avrRxo1</i> in infected leaves were confirmed by RT-PCR. All experiments were repeated three times with similar results.</p

Analysis the contribution of C- and N-terminus to the 3 function of AvrRxo1.

<p>(A) The phenotype of interactions between maize lines B73 and deletion mutants of AvrRxo1. Infiltration of B73 with PXO99^A containing <i>avrRxo1</i> gene results in HR at 2 dpi. PXO99^A (pHM1) as negative control. Fragments AvrRxo1(17–421), AvrRxo1(52–421), AvrRxo1(109–421), AvrRxo1(140–421), AvrRxo1(186–421), AvrRxo1(1–159), AvrRxo1(1–193), AvrRxo1(1–278), AvrRxo1(1–373), AvrRxo1(1–412), AvrRxo1(1–417), and AvrRxo1(1–418), abolished induce HR on B73, while fragments AvrRxo1(1–419) and AvrRxo1(1–420) produce HR on B73 at 2 dpi. (B) Suppressions of non-host HR by deletion mutants of AvrRxo1 in <i>N. benthamiana</i>. Infiltration of <i>N. benthamiana</i> with PXO99^A (pHM:avrRxo1) (1) and PXO99^A (pHM1) (2) as control. Fragments AvrRxo1(17–421) (3), AvrRxo1(52–421) (4), AvrRxo1(109–421) (5), AvrRxo1(140–421) (6), AvrRxo1(186–421) (7), AvrRxo1(1–412) (8), AvrRxo1(1–417) (9), AvrRxo1(1–418) (10), AvrRxo1(1–419) (11), AvrRxo1(1–420) (12) did not suppress the non-host HR caused by PXO99^A in <i>N. benthamiana</i>. RT-PCR was used to confirm the expression of <i>avrRxo1</i> in inoculated leaves. All experiments were repeated three times with similar results.</p

Strains and plasmids used in this study.

<p>Amp^r, ampicillin resistance; CP, coat protein; Km^r, kanamycin resistance; RFP, red fluorescent protein; Rif^r, rifampicin resistance; Sm^r, streptomycin; Sp^r, spectinomycin.</p><p>Strains and plasmids used in this study.</p

Different fragments of AvrRxo1 in frame with red fluorescent protein (RFP) display different subcellular location.

<p>The transient expression of RFP-AvrRxo1(1–421) in <i>N. benthamiana</i> revealed that the RFP-AvrRxo1 fusion protein was localised to the plasma membrane. Expressing RFP-AvrRxo1 (52–421) in <i>N. benthamiana</i> revealed that the RFP fluorescence was observed both in the plasma membrane and nucleus. The RFP fluorescence of RFP-AvrRxo1(109–421) was observed only in the nucleus. Bars  = 10 µm.</p

AvrRxo1 was screened out as a suppressor of non-host resistance in <i>Nicotiana benthamiana</i>.

<p>(A) Symptoms caused by Xoo strains PXO99^A (wild type) (1), pHM1 (2), 2C10 (clone of RS105 genomic library) (3), 8E07 (clone of RS105 genomic library) (4), pHMavrRxo1_RS105 (5) and MgCl₂ (10 mM) (6) in <i>Nicotiana benthamiana</i>. Xoo strains [1×10⁸ colony-forming units (cfu)/mL] were inoculated to <i>N. benthamiana</i> (4–6 weeks old) with a needleless syringe, and symptoms were measured at 2 days post-inoculation (dpi). (B) The phenotype of interactions between maize lines B73 and five AvrRxo1 clones: pHMavrRxo1_RS105, pHMavrRxo1_RS85, pHMavrRxo1_SDAU-1, pHMavrRxo1_JSB2-24 and pHMavrRxo1_HNB8-47. Xoo strains [1×10⁸ cfu/mL] were infiltrated into B73 (4 weeks old) with a needleless syringe, and symptoms were measured at 2 dpi. Infiltration of B73 with PXO99^A containing <i>avrRxo1</i> gene results in a hypersensitive response at 2 dpi. PXO99^A and PXO99^A (pHM1) produced no reaction. (C) Symptoms caused by five AvrRxo1 clones in Xoo strains PXO99^A (1) pHM1, (2) pHMavrRxo1_RS105 (3), pHMavrRxo1_RS85 (4), pHMavrRxo1_SDAU-1 (5), pHMavrRxo1_JSB2-24 (6), pHMavrRxo1_HNB8-47 (7), and PXO61 (8) in <i>N. benthamiana</i>. All experiments were repeated three times with similar results.</p

AvrRxo1 induces cell death in <i>N. benthamiana</i> and affects cell growth in yeast.

<p>(A) Yeast growth is inhibited by expression of AvrRxo1. GS115 yeast strains carrying empty vecter pPIC3.5 or pPIC3.5:avrRxo1_RS105 were grown overnight in repressing broth (2% glucose) at 30°C. Cultures were then diluted to OD600 = 1.0, and then serial 10-fold dilutions were spotted onto resuppressing or inducing medium (1% methanol). Photographs were taken after 2 days of growth. (B) Phenotype of transient expression of <i>avrRxo1</i> from different Xoc strains in <i>N. benthamiana</i> (4–6 weeks old). Transient expression of <i>avrRxo1</i>_SDAU-1 (1), <i>avrRxo1</i>_RS105 (2) and <i>avrRxo1</i>_RS85 (5) induces <i>N. benthamiana</i> cell death. No cell death for <i>avrRxo1</i>_JSB2-24 (3) and <i>avrRxo1</i>_HNB8-47 (4) as control pGR106 (6) treatment. (C) The serine residue at the 344 position is required for toxicity of avrRxo1 in <i>N. benthamiana</i>. The transient expression of avrRxo1 from RS105 (1) induced cell death, whereas avrRxo1 from JSB2–24 (2) did not induce tobacco cell death. AvrRxo1(S344T) (3), whose serine residue at the 344 position was substituted to threonine, failed to induce cell death when transiently expressed in <i>N. benthamiana</i>. (D) The normal expression of <i>avrRxo1</i> in infected leaves were confirmed by RT-PCR. Housekeeping gene EF1a was selected to normalize the samples. Data shown are representative of three independent experiments with similar results.</p

Model of AvrRxo1 mutants function in recognision of Rxo1, suppression of nonhost resistance, and toxin.

<p>The colour boxes indicate that: grey, putative myristoylation site; red, nuclear localization sequence; green, ATP/GTP binding sites; blue, cysteine protease motif.</p

Comparison of amino acids sequences of AvrRxo1 alleles.

<p>The grey text indicates identical sequence, and the deep grey indicates putative motifs. The asterisk indicates the residue that is associated with toxicity. The secondary structures under the sequences were predicted using Jpred 3 software. H indicates the α-helix; E indicates β-strand.</p