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Type III effectors xopN and avrBS2 contribute to the virulence of Xanthomonas oryzae pv. oryzicola strain GX01
Journal Pre-proof Type III effectors xopN and avrBS2 contribute to the virulence of Xanthomonas oryzae pv. oryzicola strain GX01 Zhou-Xiang Liao, Jian-Yuan Li, Xiu-Yu Mo, Zhe Ni, Wei Jiang, Yong-Qiang He, Sheng Huang PII:
S0923-2508(19)30120-2
DOI:
https://doi.org/10.1016/j.resmic.2019.10.002
Reference:
RESMIC 3745
To appear in:
Research in Microbiology
Received Date: 9 March 2019 Revised Date:
3 October 2019
Accepted Date: 3 October 2019
Please cite this article as: Z.-X. Liao, J.-Y. Li, X.-Y. Mo, Z. Ni, W. Jiang, Y.-Q. He, S. Huang, Type III effectors xopN and avrBS2 contribute to the virulence of Xanthomonas oryzae pv. oryzicola strain GX01, Research in Microbiologoy, https://doi.org/10.1016/j.resmic.2019.10.002. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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Type III effectors xopN and avrBS2 contribute to the
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virulence of Xanthomonas oryzae pv. oryzicola strain
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GX01
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Running title: Virulence associated non-TAL type III
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effector genes in Xanthomonas oryzae pv. oryzicola
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Zhou-Xiang Liao a, Jian-Yuan Li a, Xiu-Yu Mo a, Zhe Ni a, Wei Jiang a, Yong-Qiang He
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a, b
*, Sheng Huang a *
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a
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of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning Guangxi, 530004,
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China
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b
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Guangxi University, 100 Daxue Road, Nanning Guangxi, 530004, China
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College
National Demonstration Center for Experimental Plant Science Education, College of Agriculture,
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E-mail address: [email protected] (ZX, Liao)
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[email protected] (JY, Li)
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[email protected]
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[email protected] (Z, Ni)
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[email protected] (W, Jiang)
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[email protected] (YQ, He) *Correspondence and reprints
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[email protected] (S, Huang) *Correspondence and reprints
(XY, Mo)
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Abstract
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Xanthomonas oryzae pv. oryzicola (Xoc) depends on its type III secretion system
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(T3SS) to translocate type III secreted effectors (T3SEs), including transcription
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activator-like effectors (TALEs) and non-transcription activator-like effectors
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(non-TALEs), into host cells. T3SEs can promote the colonization of Xoc and
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contribute to virulence by manipulating host cell physiology. We annotated 25 genes
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encoding non-TALEs in Xoc strain GX01, an isolate from Guangxi in the South China’s
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rice growing region. Through systematic mutagenesis of non-TALEs, we found that
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xopN, the virulence contribution of which was previously unknown for Xoc,
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significantly contributes to the virulence of Xoc GX01, as does avrBs2.
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Key words: Xanthomonas oryzae pv. oryzicola; non-TAL type III effector genes;
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virulence; avrBs2; xopN
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Introduction
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The Gram-negative bacterium Xanthomonas oryzae pv. oryzicola (Xoc) is the
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causative agent of bacterial leaf streak (BLS), one of the most destructive diseases of
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rice [1]. The pathogen enters rice leaves through stomata or wound sites and colonizes
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intercellular spaces in the mesophyll, leading to water-soaking interveinal lesions that
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develop into translucent streaks [1]. It is now well known the type III secretion system
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(T3SS) plays a key role in Xoc infection of rice [2,3]. The T3SS is a complex
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transmembrane structure that can deliver type three secreted effectors (T3SEs) into host
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cells [4] where they can induce effector-triggered susceptibility (ETS), causing disease,
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or effector triggered immunity (ETI), initiating resistance [4]. T3SEs in Xanthomonas
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are divided into two major groups – the transcription activator-like effectors (TALEs)
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and non-transcription activator-like effectors (non-TALEs) – depending on protein
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structure and function [5]. Xoc harbors many non-TALEs and TALEs, but only a few
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of these have been demonstrated to make major contributions to virulence in
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Xanthomonas pathosystems [6,7], among them the non-TALEs avrBs2 and xopN [8–
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12]. Interestingly, a recent study in which genes encoding non-TALES of the Chinese
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strain RS105 established that only an avrBs2 mutant strain, but not a xopN mutant
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strain was significantly impaired in virulence [13], raising the question of how
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universal the contributions of “known” major effectors are. Therefore, we decided to
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investigate non-TALE contributions within another Chinese Xoc strain.
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Xoc strain GX01 was isolated from a BLS outbreak in Hezhou, Guangxi, China,
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and is highly virulent on model rice cultivar Oryza sativa L cv. Nipponbare[14,15]. To
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determine the virulence contributions of the non-TALEs in this strain, we identified
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all putative effectors in Xoc GX01 based on sequence similarity, deleted the genes
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encoding the non-TALEs individually, and assessed their contribution to virulence.
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Materials and Methods 4
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1.1 Bacteria strains and rice lines used in this study
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Xoc strain GX01 is a rifampicin-resistant spontaneous mutant of strain LT4, which
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was isolated from the rice leaf with typical BLS symptoms in Liantang Town in Hezhou
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City in Guangxi, in the central area of South China’s rice growing region [14]. The
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genome data of Xoc GX01 is available on GenBank (CP043403). Oryza sativa L. cv.
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Nipponbare was used in this study as a susceptible host of Xoc GX01.
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1.2 Bacteria growth
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Xoc strains were grown in the rich medium nutrient broth (NB) or on nutrient agar
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(NA) plates at 28°C [16]. GX01was routinely cultured with 50 µg/ml rifampicin.
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Strains containing plasmids were grown with the appropriate antibiotics to maintain
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selection for them.
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1.3 Homologous-recombination based gene deletion and
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insertional gene mutagenesis To
generate
non-TAL
effector
mutants,
we
employed
a
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homologous-recombination based gene knock out technique. Briefly, for gene deletion,
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two flanking fragments surrounding the target gene ORF were amplified from the Xoc
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genome using the primer sets listed in Supplementary Table S1. Each pair of flanking
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fragments for each target were cloned into the multiple cloning site (MCS) of
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pK18mobSacB. The resulting recombinant plasmids were introduced into GX01 via
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triparental conjugation. Kan-resistant merodiploid transconjugants were selected, and
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then were grown on 10% sucrose to counter-select for plasmid integration, resulting in
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a marker-free gene-deletion mutant that could be identified by colony PCR. For
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insertional gene mutagenesis, a ~200 bp sequence inside the start codon of the target
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gene was selected and amplified from the Xoc genome. The resulting fragments were
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cloned into the MCS of pK18mob and introduced into GX01 via triparental conjugation. 5
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The Kan-resistant transconjugants were selected and the insertion was confirmed by
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colony PCR.
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1.4 Mutant complementation
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To complement each mutant in trans, we amplified the target gene with ORF and
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cloned it into the MCS in pLARFJ [17]. The resulting plasmid was introduced into
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designated mutants via triparental conjugation. Several Tet-resistant transconjugants
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were selected, and the presence of the complementation plasmid was confirmed by
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colony PCR.
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1.5 Virulence assay
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Rice seeds were germinated by submersion in water followed by incubation in the
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dark for two days at 37°C. Five germinated seeds were planted in a small pot containing
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autoclaved soil. The plants were grown in the greenhouse under a 14/10-h light/dark
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cycle with a temperature controlled between 28°C and 30°C till they reached tillering
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stage (about 30 to 45 days).
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To prepare inoculum, Xoc strains were grown for 24 h at 28°C in NB medium, and
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the cells were collected by centrifugation and re-suspended in DI water. The
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concentration of the Xoc suspension was adjusted to O.D.600 = 0.5 (approximately 5.0 ×
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107 CFU/mL) prior to infiltration.
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Selected rice plants were inoculated with the Xoc suspension using syringe
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infiltration [18]. For each plant, the last two leaves were picked and infiltrated 1/3 of
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the way down from the leaf tip. The infiltrated plants were kept in a greenhouse and
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water soaked lesions were measured after 14 days.
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Results and discussion 6
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We identified 25 putative non-TALEs in Xoc GX01 (Table 1 and supplementary
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Table S2) by BLASTn using a database of known Xanthomonas effectors
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(http://www.Xanthomonas.org). The majority of the non-TALEs in Xoc GX01 were
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conserved with Xoc model strain BLS256 [7], and another Chinese local Xoc strain
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RS105 [13]. Among these non-TALE genes, 20 of them are preceded by by a
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plant-inducible promoter (PIP) box-like sequence [17] upstream of their coding
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sequence (Table 1) follow by a positioned -10 box-like sequence, according GX01
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genome data. Eight genes have a “perfect” PIP box, a TTCGB-(N15)-TTCGB motif
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(Table 1) (where “N” refers to any nucleic acid bases, while “B” refers to a C, T or G,
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but not A), and the others have a similar TTCGB-(N9~25)-TTCGB-(N30-32) motif.
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The presence of PIP boxes and similar motifs suggested that these non-TALE gene’s
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transcription might be regulated by the T3SS activator HrpX [17]. Meanwhile, we also
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annotated all 27 TALEs in Xoc GX01, including 2 truncTALEs that lack of acidic
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activation domain (supplementary Table S3). There are 4 TALEs present only in
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GX01 and absent in BLS256 and RS105, while the other 21 TALEs have homologues
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in BLS256 or RS105 (supplementary Table S3).
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Using homologous-recombination based gene deletion, we successfully obtained
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23 non-TALE mutants. An xopAJ insertion mutant named nKxopAJ was also
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constructed. For unknown reasons, we could not mutate xopK by either deletion or
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insertion, with several attempts. Plant assays of the non-TALE mutants indicated that
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both avrBs2 and xopN mutants gave significantly shorter lesion at 14 days post
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inoculation than did the wild-type strain (Fig 1), while the other 22 non-TALE mutants
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did not show significant differences. To confirm the contributions of avrBs2 and xopN
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to virulence, we complemented their mutants. The plant assays demonstrated that both
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complementations restored impaired virulence of the mutants (Fig 2). This result
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demonstrates that avrBs2 and xopN contribute to virulence in Xoc GX01 individually.
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In a previous study, Li et al., (2015 ) found only avrBs2 but not xopN was required for
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full virulence in Xoc RS105 [13]. Compare to RS105, the amino acid sequence of 7
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XopN in GX01 have only one aa difference at Q704H, which is unlikely to result any
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meaningful functional difference. This suggests non-TALEs may have different
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contributions to virulence in different genomic backgrounds. XopN may play a more
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important role in Nipponbare compare to Jiangang 30, the rice cultivar being used in
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that study. On the other hand, one cannot exclude that the contribution of xopN to
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virulence is masked in strain RS105, since RS105 is harboring six genes encoding
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TALEs not present in strain GX01 and these additional genes may be
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functionally-redundant with xopN. Furthermore, a double deletion mutant of both
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avrBs2 and xopN was constructed. The virulence assay showed that the double mutant
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caused shorter lesions than the single avrBs2 or xopN knockout mutants (Fig 2). This
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suggests that avrBs2 and xopN have a cumulative effect on bacterial virulence.
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Both avrBs2 and xopN are conserved in most xanthomonads. Their involvement
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in bacterial virulence has been reported in Xanthomonas campestris pv. campestris
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(Xcc), Xanthomonas campestris pv. vesicatoria (Xcv), Xanthomonas axonopodis pv.
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manihotis (Xam), and Xanthomonas axonopodis pv. punicae (Xap) and the mechanism
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by which they contribute to disease has been the subject of significant attention [8–12].
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avrBs2 was also reported to be involved in virulence in Xoc [13], and xopN in
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Xanthomonas oryzae pv. oryzae (Xoo) [19]. Previous studies showed that AvrBs2 was
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able to suppress pathogen-associated molecular pattern triggered immunity (PTI) in
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planta
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glycelophodiesterase domain which was related to virulence [9], but the mechanism
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by which AvrBs2 suppresses PTI remains unknown. Unlike in Xoc RS105, xopN
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contributes to the virulence of GX01 [13], which is consistent with the major
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virulence role of its orthologs in Xcc, Xcv, and Xoo strains. XopN is thought to
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interfere with immune signaling involved in plant innate immunity in Xcv [10],
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reducing the plant callus deposition in both Xoo [19] and Xam [12], and weakening
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host reactive oxygen species production in Xap [11]. All these studies suggest that
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both avrBs2 and xopN play crucial roles in suppressing immune responses of host
[12,13].
A
biochemical
function
8
study
showed
AvrBs2
had
a
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plants, thus allowing disease development. In summary, we annotated 25 non-TALE genes in Xoc GX01, a Chinese Xoc
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strain, and mutated 24 of them. We discovered that, dissimilar to Chinese Xoc strain
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RS105, both avrBs2 and xopN are required for the full virulence of GX01, and their
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effect is additive. Mutation of the other 22 non-TALEs did not significantly alter
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bacterial virulence in our study, but since functional redundancy can mask the
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contributions of individual effectors, further investigations are required to determine
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their roles in pathogenicity.
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Conflict of interest The authors have no actual or potential conflict of interest to disclose.
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Funding This research is sponsored by the National Key R&D Program of China
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(2018YFD0200300),
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(31860032,31660505), the Ba Gui Scholar Program of Guangxi Zhuang Autonomous
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Region of China (2014A002) and the 100 Talent Program for Colleges and
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Universities in Guangxi to Sheng Huang
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Figure Legends
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Figure 1. Virulence assays of Xanthomonas oryzae pv. oryzicola GX01 type III
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secreted non-TAL effectors mutants. (A) Results showed rice leave in tillering stage
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inoculated with non-TALE knock out mutants of GX01. The T3SE mutants ∆avrBs2
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and ∆xopN had shorter lesions 14 days post inoculation, compared with wild-type.
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Values are the mean lesion length ± standard deviation (SD) from three repeats, each
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with 20 lesions. “**” represents marked values are significantly different from wild
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type in a Student's t-test followed by Šidák correction, with α = 0.01. (B)
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Representative lesions caused by each mutant.
256 257
Figure 2. avrBs2 and xopN are necessary for the full pathogenicity of
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Xanthomonas oryzae pv. oryzicola GX01. (A) Virulence assays show that
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complemented strains ∆avrBs2/pLARFJ::avrBs2 and ∆xopN/pLARFJ::∆xopN have
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restored virulence. ∆avrBs2∆xopN, an avrBs2 and xopN double knockout mutant,
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caused shorter lesions compared to avrBs2 or xopN single knockout mutants. Values
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are the mean lesion length ± standard deviation (SD) from three repeats, each with 10
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lesions. Values with the same letter are not significantly different after the
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Ryan-Einot-Gabriel-Welsh F (REGWF) procedure, with α = 0.05. (B) Representative
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lesions caused by each strain.
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12
Table 1. PIP-Boxes of Non-TAL effectors of Xanthomonas oryzae pv. oryzicola GX01 Gene ID Gene name PIP-box and -10 motifa TTCGCATCCCGGTGGCATATTTCGCCTCGGGTGCGAATTGTCATATTTCATGCGGTCTACTGA XOCgx_4849 avrBs2 TTCGTCGTCCCAGCCAGGACTTCGGTGAGGCGAAGCAAATTGGAGAGCTACTCGCCAAACT XOCgx_2377 xopAA TTCGCACAGGTCGTCGGCGCTTCGTATTGAAGAAGTCGCTCGCGGGCAGATTGACTAAATGT XOCgx_0233 xopAD TTCGTTCGCTGCTCATATAGTTCGTAAGGTGTGCGAGTTGCGATTCGAATCGCTCTTAATGT XOCgx_0675 xopAJ TTCGTGTATGGCGCGGTTGGTTCGCCGCGCTCCGTTCGGGTGCGTATTTCATAGCGAAACAATAT XOCgx_0272 xopF1 TTCGTTTACAGAATCGCTTGTTCGTCGCAGCAAGACTGGCTGGAGAGGACAGGCGCACCAT XOCgx_1511 xopK TTCGCCAGGATAAAGATGACTTCGCCCCAAGGGCGTCGTGCTTAGCGTGGCATACCGATCAT XOCgx_1505 xopL TTCGGACTGCGCGACGCCGGTTCGCCCCCCGTCTTCGGCACCGCAATGGGATCGCTACGAT XOCgx_0115 xopR TTCGCAAGTTCTGCAGCTTTTTCGGCTGGGTGGGAGCGTTTTTGGCCAGGGTCTGTCAATTT XOCgx_2374 xopU TTCGCCTATTGCGCGGAGCATTCGCAAAACGAACCCTCCCGCCTCAGGCAACGCGACATCCT XOCgx_1283 xopY TTCGCGCAGGCGAACGCGATTTCTGCCAGTGCGCGCGGACACGAGATCGCTTTCGGGAAAGT XOCgx_4122 xopI TTCGGATGCAGCGACGTCAGTTCTGTTGTCCGGCAGAGGTGGCCCCGGCAGTTTGATTCAAT XOCgx_4624 xopN TTCGTCGCCGCACACAGGAATTCAGCACGCCGCCCTGGCGCTGCGCTGGACGTGGGTAACGT XOCgx_4891 xopQ TTCTGCTTTCAGTGCAACATTTCGCACTTGCCGCATGCTGCGGCTGCGCCTGTTCCGATCAT XOCgx_4341 xopX TTCTCTTTCCAAGCGACCACTTCGCGCAGCTGCAATGCCTCGAACGCGCCGCGCAATATTGT XOCgx_2461 xopZ1 TTCGCTTTCCAGCCCCATCGTTTCGGCAGCGGGCGCGTCTTCATGCAGGGGCGATGCGAGGAT XOCgx_0801 xopAK TTCGGCTCCAAAGTCATTCGGAATGGTGGAAAATTTTTCCTCCTGCGCCCACTGAGT XOCgx_3839 xopO TTCGCCAAGCGATACAGCCTCTTCACTTCGATGCGGCGATGGCAGTCGCATCGCATTAGCCT XOCgx_0265 xopAE TTCTCCGGGCACGGCGTTAGCATCTTGCGTCTTCACTGGGCAATGAATGACTCACATGGGTAACTG XOCgx_4518 xopAF TTCGCGTTAGCCACAGCTTCGATCGTGCAACCAGCATGCGTATCTGCGCAATGT XOCgx_3671 xopC2 TTCTGCAAGGCAGCGAAGGCTTTCGCCAAATCGCACATCGATTCTGCTGTGAGCGGTATGCA XOCgx_4305 xopV TTCAGGCCTCGTTGTTCAGATCTTCCTTATGTTGCCAAGTTGCGGCGTGGTGTTTAAATCATTTT XOCgx_4471 xopW N.A XOCgx_3272 xopAB N.A XOCgx_3675 xopP-1 N.A XOCgx_3674 xopP-2 a. PIP-box and -10 motifs are shown in bold. Underlined bases mean they mismatch standard PIP-box or -10 motifs. b. Too far away from ORF; may not functional. c. PIP-box is upstream of neighboring gene hpa3; xopF1 may be co-transcribed with hpa3 and share the same PIP-box.
distance to ORF 27 700b 6 52 682c 29 33 27 16 26 54 202 187 52 99 31 52 274 193 67 49 23 N.A N.A N.A
S0923-2508(19)30120-2
DOI:
https://doi.org/10.1016/j.resmic.2019.10.002
Reference:
RESMIC 3745
To appear in:
Research in Microbiology
Received Date: 9 March 2019 Revised Date:
3 October 2019
Accepted Date: 3 October 2019
Please cite this article as: Z.-X. Liao, J.-Y. Li, X.-Y. Mo, Z. Ni, W. Jiang, Y.-Q. He, S. Huang, Type III effectors xopN and avrBS2 contribute to the virulence of Xanthomonas oryzae pv. oryzicola strain GX01, Research in Microbiologoy, https://doi.org/10.1016/j.resmic.2019.10.002. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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Type III effectors xopN and avrBS2 contribute to the
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virulence of Xanthomonas oryzae pv. oryzicola strain
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GX01
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Running title: Virulence associated non-TAL type III
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effector genes in Xanthomonas oryzae pv. oryzicola
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Zhou-Xiang Liao a, Jian-Yuan Li a, Xiu-Yu Mo a, Zhe Ni a, Wei Jiang a, Yong-Qiang He
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a, b
*, Sheng Huang a *
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a
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of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning Guangxi, 530004,
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China
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b
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Guangxi University, 100 Daxue Road, Nanning Guangxi, 530004, China
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College
National Demonstration Center for Experimental Plant Science Education, College of Agriculture,
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E-mail address: [email protected] (ZX, Liao)
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[email protected] (JY, Li)
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[email protected]
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[email protected] (Z, Ni)
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[email protected] (W, Jiang)
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[email protected] (YQ, He) *Correspondence and reprints
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[email protected] (S, Huang) *Correspondence and reprints
(XY, Mo)
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Abstract
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Xanthomonas oryzae pv. oryzicola (Xoc) depends on its type III secretion system
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(T3SS) to translocate type III secreted effectors (T3SEs), including transcription
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activator-like effectors (TALEs) and non-transcription activator-like effectors
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(non-TALEs), into host cells. T3SEs can promote the colonization of Xoc and
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contribute to virulence by manipulating host cell physiology. We annotated 25 genes
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encoding non-TALEs in Xoc strain GX01, an isolate from Guangxi in the South China’s
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rice growing region. Through systematic mutagenesis of non-TALEs, we found that
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xopN, the virulence contribution of which was previously unknown for Xoc,
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significantly contributes to the virulence of Xoc GX01, as does avrBs2.
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Key words: Xanthomonas oryzae pv. oryzicola; non-TAL type III effector genes;
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virulence; avrBs2; xopN
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Introduction
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The Gram-negative bacterium Xanthomonas oryzae pv. oryzicola (Xoc) is the
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causative agent of bacterial leaf streak (BLS), one of the most destructive diseases of
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rice [1]. The pathogen enters rice leaves through stomata or wound sites and colonizes
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intercellular spaces in the mesophyll, leading to water-soaking interveinal lesions that
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develop into translucent streaks [1]. It is now well known the type III secretion system
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(T3SS) plays a key role in Xoc infection of rice [2,3]. The T3SS is a complex
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transmembrane structure that can deliver type three secreted effectors (T3SEs) into host
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cells [4] where they can induce effector-triggered susceptibility (ETS), causing disease,
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or effector triggered immunity (ETI), initiating resistance [4]. T3SEs in Xanthomonas
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are divided into two major groups – the transcription activator-like effectors (TALEs)
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and non-transcription activator-like effectors (non-TALEs) – depending on protein
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structure and function [5]. Xoc harbors many non-TALEs and TALEs, but only a few
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of these have been demonstrated to make major contributions to virulence in
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Xanthomonas pathosystems [6,7], among them the non-TALEs avrBs2 and xopN [8–
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12]. Interestingly, a recent study in which genes encoding non-TALES of the Chinese
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strain RS105 established that only an avrBs2 mutant strain, but not a xopN mutant
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strain was significantly impaired in virulence [13], raising the question of how
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universal the contributions of “known” major effectors are. Therefore, we decided to
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investigate non-TALE contributions within another Chinese Xoc strain.
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Xoc strain GX01 was isolated from a BLS outbreak in Hezhou, Guangxi, China,
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and is highly virulent on model rice cultivar Oryza sativa L cv. Nipponbare[14,15]. To
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determine the virulence contributions of the non-TALEs in this strain, we identified
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all putative effectors in Xoc GX01 based on sequence similarity, deleted the genes
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encoding the non-TALEs individually, and assessed their contribution to virulence.
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Materials and Methods 4
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1.1 Bacteria strains and rice lines used in this study
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Xoc strain GX01 is a rifampicin-resistant spontaneous mutant of strain LT4, which
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was isolated from the rice leaf with typical BLS symptoms in Liantang Town in Hezhou
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City in Guangxi, in the central area of South China’s rice growing region [14]. The
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genome data of Xoc GX01 is available on GenBank (CP043403). Oryza sativa L. cv.
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Nipponbare was used in this study as a susceptible host of Xoc GX01.
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1.2 Bacteria growth
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Xoc strains were grown in the rich medium nutrient broth (NB) or on nutrient agar
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(NA) plates at 28°C [16]. GX01was routinely cultured with 50 µg/ml rifampicin.
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Strains containing plasmids were grown with the appropriate antibiotics to maintain
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selection for them.
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1.3 Homologous-recombination based gene deletion and
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insertional gene mutagenesis To
generate
non-TAL
effector
mutants,
we
employed
a
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homologous-recombination based gene knock out technique. Briefly, for gene deletion,
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two flanking fragments surrounding the target gene ORF were amplified from the Xoc
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genome using the primer sets listed in Supplementary Table S1. Each pair of flanking
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fragments for each target were cloned into the multiple cloning site (MCS) of
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pK18mobSacB. The resulting recombinant plasmids were introduced into GX01 via
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triparental conjugation. Kan-resistant merodiploid transconjugants were selected, and
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then were grown on 10% sucrose to counter-select for plasmid integration, resulting in
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a marker-free gene-deletion mutant that could be identified by colony PCR. For
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insertional gene mutagenesis, a ~200 bp sequence inside the start codon of the target
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gene was selected and amplified from the Xoc genome. The resulting fragments were
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cloned into the MCS of pK18mob and introduced into GX01 via triparental conjugation. 5
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The Kan-resistant transconjugants were selected and the insertion was confirmed by
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colony PCR.
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1.4 Mutant complementation
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To complement each mutant in trans, we amplified the target gene with ORF and
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cloned it into the MCS in pLARFJ [17]. The resulting plasmid was introduced into
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designated mutants via triparental conjugation. Several Tet-resistant transconjugants
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were selected, and the presence of the complementation plasmid was confirmed by
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colony PCR.
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1.5 Virulence assay
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Rice seeds were germinated by submersion in water followed by incubation in the
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dark for two days at 37°C. Five germinated seeds were planted in a small pot containing
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autoclaved soil. The plants were grown in the greenhouse under a 14/10-h light/dark
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cycle with a temperature controlled between 28°C and 30°C till they reached tillering
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stage (about 30 to 45 days).
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To prepare inoculum, Xoc strains were grown for 24 h at 28°C in NB medium, and
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the cells were collected by centrifugation and re-suspended in DI water. The
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concentration of the Xoc suspension was adjusted to O.D.600 = 0.5 (approximately 5.0 ×
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107 CFU/mL) prior to infiltration.
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Selected rice plants were inoculated with the Xoc suspension using syringe
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infiltration [18]. For each plant, the last two leaves were picked and infiltrated 1/3 of
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the way down from the leaf tip. The infiltrated plants were kept in a greenhouse and
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water soaked lesions were measured after 14 days.
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Results and discussion 6
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We identified 25 putative non-TALEs in Xoc GX01 (Table 1 and supplementary
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Table S2) by BLASTn using a database of known Xanthomonas effectors
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(http://www.Xanthomonas.org). The majority of the non-TALEs in Xoc GX01 were
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conserved with Xoc model strain BLS256 [7], and another Chinese local Xoc strain
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RS105 [13]. Among these non-TALE genes, 20 of them are preceded by by a
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plant-inducible promoter (PIP) box-like sequence [17] upstream of their coding
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sequence (Table 1) follow by a positioned -10 box-like sequence, according GX01
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genome data. Eight genes have a “perfect” PIP box, a TTCGB-(N15)-TTCGB motif
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(Table 1) (where “N” refers to any nucleic acid bases, while “B” refers to a C, T or G,
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but not A), and the others have a similar TTCGB-(N9~25)-TTCGB-(N30-32) motif.
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The presence of PIP boxes and similar motifs suggested that these non-TALE gene’s
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transcription might be regulated by the T3SS activator HrpX [17]. Meanwhile, we also
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annotated all 27 TALEs in Xoc GX01, including 2 truncTALEs that lack of acidic
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activation domain (supplementary Table S3). There are 4 TALEs present only in
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GX01 and absent in BLS256 and RS105, while the other 21 TALEs have homologues
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in BLS256 or RS105 (supplementary Table S3).
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Using homologous-recombination based gene deletion, we successfully obtained
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23 non-TALE mutants. An xopAJ insertion mutant named nKxopAJ was also
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constructed. For unknown reasons, we could not mutate xopK by either deletion or
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insertion, with several attempts. Plant assays of the non-TALE mutants indicated that
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both avrBs2 and xopN mutants gave significantly shorter lesion at 14 days post
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inoculation than did the wild-type strain (Fig 1), while the other 22 non-TALE mutants
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did not show significant differences. To confirm the contributions of avrBs2 and xopN
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to virulence, we complemented their mutants. The plant assays demonstrated that both
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complementations restored impaired virulence of the mutants (Fig 2). This result
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demonstrates that avrBs2 and xopN contribute to virulence in Xoc GX01 individually.
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In a previous study, Li et al., (2015 ) found only avrBs2 but not xopN was required for
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full virulence in Xoc RS105 [13]. Compare to RS105, the amino acid sequence of 7
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XopN in GX01 have only one aa difference at Q704H, which is unlikely to result any
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meaningful functional difference. This suggests non-TALEs may have different
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contributions to virulence in different genomic backgrounds. XopN may play a more
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important role in Nipponbare compare to Jiangang 30, the rice cultivar being used in
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that study. On the other hand, one cannot exclude that the contribution of xopN to
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virulence is masked in strain RS105, since RS105 is harboring six genes encoding
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TALEs not present in strain GX01 and these additional genes may be
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functionally-redundant with xopN. Furthermore, a double deletion mutant of both
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avrBs2 and xopN was constructed. The virulence assay showed that the double mutant
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caused shorter lesions than the single avrBs2 or xopN knockout mutants (Fig 2). This
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suggests that avrBs2 and xopN have a cumulative effect on bacterial virulence.
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Both avrBs2 and xopN are conserved in most xanthomonads. Their involvement
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in bacterial virulence has been reported in Xanthomonas campestris pv. campestris
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(Xcc), Xanthomonas campestris pv. vesicatoria (Xcv), Xanthomonas axonopodis pv.
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manihotis (Xam), and Xanthomonas axonopodis pv. punicae (Xap) and the mechanism
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by which they contribute to disease has been the subject of significant attention [8–12].
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avrBs2 was also reported to be involved in virulence in Xoc [13], and xopN in
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Xanthomonas oryzae pv. oryzae (Xoo) [19]. Previous studies showed that AvrBs2 was
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able to suppress pathogen-associated molecular pattern triggered immunity (PTI) in
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planta
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glycelophodiesterase domain which was related to virulence [9], but the mechanism
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by which AvrBs2 suppresses PTI remains unknown. Unlike in Xoc RS105, xopN
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contributes to the virulence of GX01 [13], which is consistent with the major
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virulence role of its orthologs in Xcc, Xcv, and Xoo strains. XopN is thought to
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interfere with immune signaling involved in plant innate immunity in Xcv [10],
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reducing the plant callus deposition in both Xoo [19] and Xam [12], and weakening
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host reactive oxygen species production in Xap [11]. All these studies suggest that
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both avrBs2 and xopN play crucial roles in suppressing immune responses of host
[12,13].
A
biochemical
function
8
study
showed
AvrBs2
had
a
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plants, thus allowing disease development. In summary, we annotated 25 non-TALE genes in Xoc GX01, a Chinese Xoc
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strain, and mutated 24 of them. We discovered that, dissimilar to Chinese Xoc strain
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RS105, both avrBs2 and xopN are required for the full virulence of GX01, and their
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effect is additive. Mutation of the other 22 non-TALEs did not significantly alter
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bacterial virulence in our study, but since functional redundancy can mask the
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contributions of individual effectors, further investigations are required to determine
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their roles in pathogenicity.
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Conflict of interest The authors have no actual or potential conflict of interest to disclose.
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Funding This research is sponsored by the National Key R&D Program of China
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(2018YFD0200300),
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(31860032,31660505), the Ba Gui Scholar Program of Guangxi Zhuang Autonomous
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Region of China (2014A002) and the 100 Talent Program for Colleges and
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Universities in Guangxi to Sheng Huang
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Figure Legends
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Figure 1. Virulence assays of Xanthomonas oryzae pv. oryzicola GX01 type III
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secreted non-TAL effectors mutants. (A) Results showed rice leave in tillering stage
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inoculated with non-TALE knock out mutants of GX01. The T3SE mutants ∆avrBs2
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and ∆xopN had shorter lesions 14 days post inoculation, compared with wild-type.
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Values are the mean lesion length ± standard deviation (SD) from three repeats, each
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with 20 lesions. “**” represents marked values are significantly different from wild
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type in a Student's t-test followed by Šidák correction, with α = 0.01. (B)
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Representative lesions caused by each mutant.
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Figure 2. avrBs2 and xopN are necessary for the full pathogenicity of
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Xanthomonas oryzae pv. oryzicola GX01. (A) Virulence assays show that
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complemented strains ∆avrBs2/pLARFJ::avrBs2 and ∆xopN/pLARFJ::∆xopN have
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restored virulence. ∆avrBs2∆xopN, an avrBs2 and xopN double knockout mutant,
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caused shorter lesions compared to avrBs2 or xopN single knockout mutants. Values
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are the mean lesion length ± standard deviation (SD) from three repeats, each with 10
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lesions. Values with the same letter are not significantly different after the
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Ryan-Einot-Gabriel-Welsh F (REGWF) procedure, with α = 0.05. (B) Representative
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lesions caused by each strain.
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Table 1. PIP-Boxes of Non-TAL effectors of Xanthomonas oryzae pv. oryzicola GX01 Gene ID Gene name PIP-box and -10 motifa TTCGCATCCCGGTGGCATATTTCGCCTCGGGTGCGAATTGTCATATTTCATGCGGTCTACTGA XOCgx_4849 avrBs2 TTCGTCGTCCCAGCCAGGACTTCGGTGAGGCGAAGCAAATTGGAGAGCTACTCGCCAAACT XOCgx_2377 xopAA TTCGCACAGGTCGTCGGCGCTTCGTATTGAAGAAGTCGCTCGCGGGCAGATTGACTAAATGT XOCgx_0233 xopAD TTCGTTCGCTGCTCATATAGTTCGTAAGGTGTGCGAGTTGCGATTCGAATCGCTCTTAATGT XOCgx_0675 xopAJ TTCGTGTATGGCGCGGTTGGTTCGCCGCGCTCCGTTCGGGTGCGTATTTCATAGCGAAACAATAT XOCgx_0272 xopF1 TTCGTTTACAGAATCGCTTGTTCGTCGCAGCAAGACTGGCTGGAGAGGACAGGCGCACCAT XOCgx_1511 xopK TTCGCCAGGATAAAGATGACTTCGCCCCAAGGGCGTCGTGCTTAGCGTGGCATACCGATCAT XOCgx_1505 xopL TTCGGACTGCGCGACGCCGGTTCGCCCCCCGTCTTCGGCACCGCAATGGGATCGCTACGAT XOCgx_0115 xopR TTCGCAAGTTCTGCAGCTTTTTCGGCTGGGTGGGAGCGTTTTTGGCCAGGGTCTGTCAATTT XOCgx_2374 xopU TTCGCCTATTGCGCGGAGCATTCGCAAAACGAACCCTCCCGCCTCAGGCAACGCGACATCCT XOCgx_1283 xopY TTCGCGCAGGCGAACGCGATTTCTGCCAGTGCGCGCGGACACGAGATCGCTTTCGGGAAAGT XOCgx_4122 xopI TTCGGATGCAGCGACGTCAGTTCTGTTGTCCGGCAGAGGTGGCCCCGGCAGTTTGATTCAAT XOCgx_4624 xopN TTCGTCGCCGCACACAGGAATTCAGCACGCCGCCCTGGCGCTGCGCTGGACGTGGGTAACGT XOCgx_4891 xopQ TTCTGCTTTCAGTGCAACATTTCGCACTTGCCGCATGCTGCGGCTGCGCCTGTTCCGATCAT XOCgx_4341 xopX TTCTCTTTCCAAGCGACCACTTCGCGCAGCTGCAATGCCTCGAACGCGCCGCGCAATATTGT XOCgx_2461 xopZ1 TTCGCTTTCCAGCCCCATCGTTTCGGCAGCGGGCGCGTCTTCATGCAGGGGCGATGCGAGGAT XOCgx_0801 xopAK TTCGGCTCCAAAGTCATTCGGAATGGTGGAAAATTTTTCCTCCTGCGCCCACTGAGT XOCgx_3839 xopO TTCGCCAAGCGATACAGCCTCTTCACTTCGATGCGGCGATGGCAGTCGCATCGCATTAGCCT XOCgx_0265 xopAE TTCTCCGGGCACGGCGTTAGCATCTTGCGTCTTCACTGGGCAATGAATGACTCACATGGGTAACTG XOCgx_4518 xopAF TTCGCGTTAGCCACAGCTTCGATCGTGCAACCAGCATGCGTATCTGCGCAATGT XOCgx_3671 xopC2 TTCTGCAAGGCAGCGAAGGCTTTCGCCAAATCGCACATCGATTCTGCTGTGAGCGGTATGCA XOCgx_4305 xopV TTCAGGCCTCGTTGTTCAGATCTTCCTTATGTTGCCAAGTTGCGGCGTGGTGTTTAAATCATTTT XOCgx_4471 xopW N.A XOCgx_3272 xopAB N.A XOCgx_3675 xopP-1 N.A XOCgx_3674 xopP-2 a. PIP-box and -10 motifs are shown in bold. Underlined bases mean they mismatch standard PIP-box or -10 motifs. b. Too far away from ORF; may not functional. c. PIP-box is upstream of neighboring gene hpa3; xopF1 may be co-transcribed with hpa3 and share the same PIP-box.
distance to ORF 27 700b 6 52 682c 29 33 27 16 26 54 202 187 52 99 31 52 274 193 67 49 23 N.A N.A N.A