Cell Wall Degrading Enzyme Induced Rice Innate Immune

Responses Are Suppressed by the Type 3 Secretion

System Effectors XopN, XopQ, XopX and XopZ of
Xanthomonas oryzae pv. oryzae
Dipanwita Sinha, Mahesh Kumar Gupta, Hitendra Kumar Patel¤, Ashish Ranjan, Ramesh V. Sonti*
CSIR-Centre for Cellular and Molecular Biology, Hyderabad, Andhra Pradesh, India

Abstract

Innate immune responses are induced in plants and animals through perception of Damage Associated Molecular
Patterns. These immune responses are suppressed by pathogens during infection. A number of studies have
focussed on identifying functions of plant pathogenic bacteria that are involved in suppression of Pathogen
Associated Molecular Pattern induced immune responses. In comparison, there is very little information on functions
used by plant pathogens to suppress Damage Associated Molecular Pattern induced immune responses.
Xanthomonas oryzae pv. oryzae, a gram negative bacterial pathogen of rice, secretes hydrolytic enzymes such as
LipA (Lipase/Esterase) that damage rice cell walls and induce innate immune responses. Here, we show that
Agrobacterium mediated transient transfer of the gene for XopN, a X. oryzae pv. oryzae type 3 secretion (T3S)
system effector, results in suppression of rice innate immune responses induced by LipA. A xopN- mutant of X.
oryzae pv. oryzae retains the ability to suppress these innate immune responses indicating the presence of other
functionally redundant proteins. In transient transfer assays, we have assessed the ability of 15 other X. oryzae pv.
oryzae T3S secreted effectors to suppress rice innate immune responses. Amongst these proteins, XopQ, XopX and
XopZ are suppressors of LipA induced innate immune responses. A mutation in any one of the xopN, xopQ, xopX or
xopZ genes causes partial virulence deficiency while a xopN- xopX- double mutant exhibits a greater virulence
deficiency. A xopN- xopQ- xopX- xopZ- quadruple mutant of X. oryzae pv. oryzae induces callose deposition, an
innate immune response, similar to a X. oryzae pv. oryzae T3S- mutant in rice leaves. Overall, these results indicate
that multiple T3S secreted proteins of X. oryzae pv. oryzae can suppress cell wall damage induced rice innate
immune responses.

Citation: Sinha D, Gupta MK, Patel HK, Ranjan A, Sonti RV (2013) Cell Wall Degrading Enzyme Induced Rice Innate Immune Responses Are Suppressed
by the Type 3 Secretion System Effectors XopN, XopQ, XopX and XopZ of Xanthomonas oryzae pv. oryzae. PLoS ONE 8(9): e75867. doi:10.1371/
[Link].0075867
Editor: Esteban Chaves-Olarte, Universidad de Costa Rica, Costa Rica
Received April 23, 2013; Accepted August 16, 2013; Published September 26, 2013
Copyright: © 2013 Sinha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants to RVS from the Department of Biotechnology (DBT), Government of India and the Plant-Microbe and Soil
Interactions project of the Council of Scientific and Industrial Research (CSIR). DS and MKG were supported by doctoral and post-doctoral fellowships,
respectively, from the DBT. AR was supported by a doctoral fellowship from CSIR. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: sonti@[Link]
¤ Current address: International Centre for Genetic Engineering & Biotechnology, Trieste, Italy

Introduction tissue injury that occurs during growth, stress and pathogen
entry [2]. Pattern recognition receptors (PRRs) are involved in
The innate immune systems of plants and animals are perception of PAMPs and DAMPs. In animals, recognition of
activated by the perception of danger signals in the form of either PAMPs or DAMPs activates the innate immune system
pathogen associated molecular patterns (PAMPs) and and results in various inflammatory responses [3]. In plants, the
damage-associated molecular patterns (DAMPs). PAMPs are perception of these danger signals results in the activation of
indispensable, structurally conserved molecular features that the first layer of the plant innate immune system which is
are unique to a broad class of microbes and typify ‘non-self’ termed as PAMP-triggered immunity or PTI [4,5]. Suppression
because they are not present in the host [1] whereas DAMPs of PTI appears to be a crucial attribute of plant pathogens. A
are mostly endogenous molecules which are released upon number of studies have shown that Gram negative plant

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 Suppression of Damage Induced Innate Immunity

pathogenic bacteria suppress PTI using proteins that are pathogen to cause disease. Purified preparations of enzymes
secreted into plant cells via the type 3 secretion system (T3S). LipA, ClsA and CbsA have been shown to induce innate
The gram negative bacterial genus Xanthomonas is immune responses such as callose deposition and
comprised of bacteria that cause almost ~ 400 different plant programmed cell death in rice tissues [22]. Enzyme activity is
diseases. These bacteria secrete a number of effector proteins required for induction of innate immunity as seen from the
into their host cells using the T3S. These include both TAL observation that a point mutation (S176A) in the active site of
(transcription activator-like) effectors and non-TAL effectors. the LipA protein results in loss of enzyme activity and ability to
Unlike TAL effectors which benefit xanthomonads by altering induce immune responses [23]. X. oryzae pv. oryzae can
host gene transcription, the non-TAL effectors appear to suppress cell wall damage induced innate immune responses
function in suppression of host innate immunity, presumably by using a T3S system. The T3S- mutant is itself an inducer of
interfering with signaling events. The biochemical functions of innate immunity and a T2S- T3S- double mutant is
the majority of non-TAL effectors are unknown, although a few compromised in induction of innate immunity suggesting that
of them are known to have enzymatic activity. AvrBs2 has T2S secreted proteins play an important role in induction of
putative glycerophosphoryldiester phosphodiesterase activity innate immunity during infection [22]. The X. oryzae pv. oryzae
[6] whereas XopQ is predicted to have a inosine-uridine T3S secreted effectors (T3Es) that are involved in suppression
nucleoside N-ribohydrolase activity [7]. of cell wall damage induced rice innate immunity have not yet
The role of individual non-TAL effectors of xanthomonads in been identified.
suppression of host innate immunity and promotion of virulence In this study, we tested the ability of 16 different T3S
has been examined. The non-TAL effectors of Xanthomonas secreted non-TAL effectors of X. oryzae pv. oryzae [24] to
campestris pv. vesicatoria such as XopN [8], XopJ [9], XopB suppress cell wall damage induced innate immunity in rice. We
and XopS [10] have been shown to suppress PAMP induced found that Agrobacterium mediated transient transfer of xopN
innate immune responses such as callose deposition in host gene of X. oryzae pv. oryzae suppressed cell wall damage
plants. Suppression of plant defense responses has been also induced programmed cell death in rice roots and callose
reported by X. campestris pv. vesicatoria effector XopX when deposition in rice leaves mediated by enzyme LipA. However,
expressed ectopically in Nicotiana benthamiana [11] and XopD, the ability of a xopN- mutant to suppress innate immunity
a SUMO protease, that promotes pathogen growth at later induced by LipA indicated the presence of other functionally
stages of infection in the host plant tomato [12]. In addition, redundant T3S effectors. We thus screened 15 other T3S
upon ectopic expression, X. oryzae pv. oryzae effectors XopZ secreted proteins of X. oryzae pv. oryzae and identified XopQ,
[13] and XopR [14] have been reported to suppress PAMP XopX and XopZ proteins as suppressors of LipA induced
induced callose deposition in N. benthamiana and Arabidopsis innate immunity in rice. For the remaining 12 effectors, namely
respectively. Furthermore, mutations in many of these effector XopF, XopK, XopP, XopR, XopT, XopU, XopV, XopW, XopY,
proteins renders the pathogen partially virulence deficient and XopAA, XopAB and AvrBs2, no evidence for suppressor
impaired in symptom development on their respective hosts. activity was found although it cannot be completely ruled out.
Other T3S secreted non-TAL effectors which have been This is the first report showing role of bacterial T3Es in
reported to have a role in virulence and symptom development suppression of DTI in host-pathogen interactions.
are HpaF [15], AvrXv4 [16] and AvrXccC [17]. In contrast to the
large body of work that is available on suppression of PTI, very Results
little information is available about the functions involved in
suppression of DAMP triggered innate immunity (DTI) either in XopN protein of X. oryzae pv. oryzae suppresses cell
xanthomonads or in other plant pathogens. wall damage induced programmed cell death in rice
The plant cell wall is a formidable barrier for plant pathogens. roots
Plant cell wall degradation is a key aspect of microbial plant In a previously published study of Xanthomonas T3S
pathogenesis. Plant pathogenic fungi and bacteria secrete a effectors, mutation of the xopN gene of the tomato pathogen X.
cocktail of specific hydrolytic enzymes like cellulases, campestris pv. vesicatoria was found to affect virulence [7]. It
xylanases, polygalacturonases, pectate lyases, pectin had also been suggested, based on the presence of ARM/
esterases etc. to sever different components of the plant cell HEAT repeats, that the XopN protein might modulate plant
wall [18]. However, these cell wall degrading enzymes are signal transduction by interfering with host proteins containing
double edged swords, as the cell wall degradation products these repeats. Therefore, we decided to first address the ability
that are released by their action serve as marks of infection of X. oryzae pv. oryzae XopN protein to suppress host defence
that are sensed by the plant as DAMPs and results in activation responses induced by cell wall degrading enzyme, LipA.
of potent innate immune responses [19,20]. Localized programmed cell death (PCD) is an important host
The rice pathogen, Xanthomonas oryzae pv. oryzae, defence response against pathogen attack [25]. In a previous
secretes several cell wall degrading enzymes including a study, it was shown that a PCD is induced in rice roots by
lipase/esterase (LipA), cellulase (ClsA), cellobiosidase (CbsA), treatment with X. oryzae pv. oryzae secreted hydrolytic
and xylanase (XynB), using a Type 2 secretion (T2S) system enzymes such as LipA (Lipase/esterase A) [22]. The extent of
[21–23]. Mutations in genes for each of these individual T2S PCD was assessed by staining with propidium iodide (PI) and
secreted proteins cause a reduction in X. oryzae pv. oryzae visualization under a confocal laser scanning microscope. We
virulence indicating that they are important for the ability of this cloned the xopN gene into the MCS (multiple cloning site) of

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 Suppression of Damage Induced Innate Immunity

the binary vector pUbi-C1300 [26] wherein its expression is exhibited only cell wall associated fluorescence whereas roots
under the control of the maize ubiquitin promoter. Rice roots treated with purified LipA (Figure 2B) exhibited extensive cell
treated with LipA (Figure 1B) took up PI and the PI stained death. Interestingly, pretreatment with either the xopN- (Figure
material was dispersed within the cell, indicating nuclear 2C) or wild type (Figure 2D) strains resulted in suppression of
fragmentation. Buffer (Figure 1A) treated rice roots excluded PI LipA induced cell death. Although some of the cells took up PI,
and showed only cell wall associated fluorescence. Rice roots the extent of intracellular dispersion of PI staining material was
that were pre-treated with Agrobacterium strain EHA105/pUbi- limited as compared to that observed in roots treated with LipA
xopN followed by treatment with LipA (Figure 1C) excluded PI (Figure 2B) alone. This prompted us to check if the X. oryzae
and exhibited only cell wall associated fluorescence indicating pv. oryzae xopN- mutant can also suppress innate immune
absence of any cell death. In contrast, LipA (Figure 1D) response associated callose deposition. Co-infiltration of rice
treatment caused extensive cell death in roots which had been leaves with either xopN- (Figure 2G) or wild type (Figure 2H)
pre-treated with Agrobacterium strain EHA105/pUbi (empty along with LipA resulted in lesser amounts of callose deposition
vector). as compared to infiltration with LipA (Figure 2F) alone. This
RT-PCR analysis was performed to confirm that xopN indicated that a xopN- mutant is proficient in suppression of
transcripts were expressed in rice roots treated with EHA105/ LipA induced callose deposition in rice leaves (Figure 2I).
pUbi-xopN (Figure S1). A 123 bp product was obtained after
RT-PCR with RNA isolated from rice roots treated with Transient expression of xopQ, xopX or xopZ genes of
EHA105/pUbi-xopN but was not observed in roots treated with X. oryzae pv. oryzae suppresses cell wall damage
EHA105/pUbi. Also, as expected, a 158 bp RT-PCR product of induced PCD in rice roots
the rice GAPDH gene was obtained from rice roots that were The ability of a X. oryzae pv. oryzae xopN- mutant to
pre-treated with Agrobacterium (either EHA105/pUbi-xopN or suppress innate immune responses induced by LipA indicated
EHA105/pUbi) (Figure S1A). the presence of other functionally redundant T3S effectors. We
therefore assessed the ability of 15 non-TAL T3S effectors of
XopN protein of X. oryzae pv. oryzae suppresses LipA X. oryzae pv. oryzae, from the set of previously identified X.
induced callose deposition in rice leaves oryzae pv. oryzae T3S effectors [24], to suppress LipA induced
Callose deposition is a basal defense response to strengthen PCD in rice roots. The genes for 15 of these non-TAL T3S
the cell wall and is characterized by deposition of a β-1, 3- effectors (xopF, xopK, xopP, xopQ, xopR, xopT, xopU, xopV,
glucan polymer around the site of pathogen entry [27]. Upon xopW, xopX, xopY, xopZ, xopAA, xopAB and avrBs2) were
aniline blue staining, callose deposits appear as bright spots cloned into the Agrobacterial T-DNA vector as described in
when viewed under an epifluorescence microscope. Infiltration materials and methods. Rice roots were individually pretreated
of LipA results in induction of callose deposition in rice leaves with Agrobacterium strain EHA105 carrying the genes for each
[22]. We assessed whether Agrobacterium mediated transient of these individual T3S effectors cloned into a T-DNA vector.
transfer of the xopN gene would result in suppression of LipA These roots were subsequently treated with LipA enzyme
induced callose deposition. Rice leaves, hand infiltrated with followed by PI staining and sectioning under confocal laser
purified LipA (Figure 1F) exhibited substantially higher amounts scanning microscope. As expected, rice roots treated with LipA
of callose deposits compared with leaves infiltrated with buffer + 17-β-estradiol (Est) (Figure 3B) exhibited dispersed
(Figure 1E) alone. Co-infiltration of LipA (Figure 1G) along with intracellular PI staining indicating extensive cell death whereas
Agrobacterium strain EHA105/pUbi-xopN resulted in buffer (+Est) (Figure 3A) treated roots showed only cell wall
suppression of callose deposition to a level that is observed in associated fluorescence indicating no cell death. The rice roots
buffer (Figure 1E) infiltrated leaves. Rice leaves co-infiltrated pretreated with Agrobacterium strain EHA105 carrying pMDC7-
with LipA (Figure 1H) and Agrobacterium strain EHA105/pUbi xopQ (Figure 3C), pMDC7-xopX (Figure 3E) or pMDC7-xopZ
(empty vector) exhibited higher amounts of callose deposits (Figure 3G) in the presence of Est followed by LipA treatment
that were no different from those observed in leaves infiltrated exhibited cell wall associated fluorescence with no intracellular
with LipA (Figure 1F) alone (Figure 1I). These data indicated PI staining. However, in the absence of Est, roots pretreated
that transient transfer of the xopN gene of X. oryzae pv. oryzae with these strains followed by LipA treatment showed
suppresses callose deposition that is induced by treatment with dispersed intracellular PI staining indicating extensive cell
cell wall degrading enzyme (LipA) in rice leaves. death (Figure 3D, F and H respectively). This indicates that
induction of expression of xopQ, xopX or xopZ genes is
A xopN- mutant of X. oryzae pv. oryzae retains the sufficient for suppression of LipA induced PCD. Pretreatment
ability to suppress rice innate immunity with EHA105 carrying Agrobacterial T-DNA vector with any of
A xopN gene disruption mutant of X. oryzae pv. oryzae the following genes xopF, xopK, xopP, xopR, xopT, xopU,
(xopN-) was isolated by homologous plasmid integration. We xopV, xopW, xopY, xopAA, xopAB or avrBs2 did not result in
next tested the ability of a X. oryzae pv. oryzae xopN- mutant to suppression of PCD either in the presence or absence of Est
suppress LipA induced innate immunity. To address this issue, (data not shown). Thus, only XopQ, XopX and XopZ appear to
rice roots were pre-treated with either xopN- or wild type function as suppressors of LipA induced PCD in rice roots. RT-
followed by treatment with purified LipA. The roots were then PCR analysis was performed to confirm that xopQ, xopR, xopX
stained with PI and viewed under a confocal laser scanning and xopZ transcripts were expressed in rice roots treated with
microscope. As expected, roots treated with buffer (Figure 2A) EHA105/pMDC7-xopQ/R/X/Z gene only in the presence of

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 Suppression of Damage Induced Innate Immunity

Figure 1. Agrobacterium mediated transient transfer of the xopN gene of Xanthomonas oryzae pv. oryzae suppresses
LipA induced programmed cell death in rice roots and callose deposition in rice leaves. (A) to (D) Rice roots were treated
with one of the following: buffer (A); LipA (B); pretreatment with EHA105/pUbi-xopN followed by treatment with LipA (C);
pretreatment with EHA105/pUbi followed by treatment with LipA (D). Treated roots were subsequently stained with propidium iodide
(PI) and sectioned using confocal microscopy. Internalisation of PI is indicative of defense response-associated programmed cell
death in rice roots. Scale bar measures 20µm. (E) to (H) Rice leaves were infiltrated with one of the following: buffer (E); LipA (F);
EHA105/pUbi-xopN along with LipA (G); EHA105/pUbi along with LipA (H). The treated leaves were subsequently stained with
aniline blue and visualized under an epifluorescence microscope. White dots in these pictures are indicative of callose deposition.
Scale bar measures 100µm. (I) Mean and standard deviation were calculated for number of callose deposits observed in an area of
0.60 mm2. Data were collected from at least five leaves per treatment in each experiment (three experiments indicated as ExpI,
ExpII and ExpIII) and two to three different viewing areas from the infiltrated region of each leaf. Statistically significant differences
at P < 0.05 (Student’s two-tailed t test for independent means) were obtained from leaves infiltrated with EHA105/pUbi-xopN along
with LipA as compared to leaves treated with LipA alone. These differences were not observed in leaves infiltrated with EHA105/
pUbi along with LipA as compared to leaves treated with LipA alone. WT Xoo = wild type X. oryzae pv. oryzae. WT Xoo = wild type
X. oryzae pv. oryzae.
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 Suppression of Damage Induced Innate Immunity

Figure 2. A xopN- mutant of Xanthomonas oryzae pv. oryzae is proficient in suppression of LipA induced cell death in rice
roots and callose deposition in rice leaves. (A) to (D) Rice roots were treated with one of the following: buffer (A); LipA (B);
pretreatment with xopN- mutant followed by treatment with LipA (C); pretreatment with wild type followed by treatment with LipA (D).
Treated roots were subsequently stained with propidium iodide (PI) and viewed under a confocal microscope. Internalisation of PI is
indicative of defense response-associated programmed cell death. Scale bar measures 20µm. (E) to (H) Rice leaves were infiltrated
with one of the following: buffer (E); LipA (F); xopN- mutant along with LipA (G); wild type along with LipA (H). The infiltrated leaves
were stained for callose and visualised under an epifluorescence microscope. Callose deposits appeared as sharp white spots in
the viewed field. Scale bar measures 100µm. (I) Mean and standard deviation were calculated for number of callose deposits from
an area of 0.60 mm2. Data were collected from at least five leaves per treatment in each experiment (three experiments indicated as
ExpI, ExpII and ExpIII) and two to three different viewing areas from the infiltrated region of each leaf. Statistically significant
differences at P < 0.05 (Student’s two-tailed t test for independent means) were observed in values obtained from leaves infiltrated
with xopN- mutant (or wild type) along with LipA as compared to leaves infiltrated with LipA alone. WT Xoo = wild type X. oryzae pv.
oryzae.
doi: 10.1371/[Link].0075867.g002

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 Suppression of Damage Induced Innate Immunity

estradiol (Figure S1B). RT-PCR analysis also indicated that the

xopP gene is expressed in rice roots following transient assays
using EHA105/pUbi-xopP (Figure S1A). The xopP and xopR
genes were chosen for expression analysis as representatives
of T3S effectors which do not suppress cell death in the
transient transfer assay. The results indicate that, at least for
xopP and xopR and possibly for the other T3S effectors that do
not suppress cell death, the absence of suppression is not due
to lack of expression of the gene in rice roots during transient
assays.

Transient expression of xopQ, xopX or xopZ genes of

X. oryzae pv. oryzae suppresses cell wall damage
induced callose deposition in rice leaves
We then proceeded to assess whether transient transfer of
any of these 15 non-TAL effectors would suppress LipA
induced callose deposition in rice leaves. Co-infiltration of LipA
along with Agrobacterium strain EHA105 carrying pMDC7-
xopQ (Figure 4C), pMDC7-xopX (Figure 4E) or pMDC7-xopZ
(Figure 4G) in the presence of Est resulted in significantly less
callose deposition than that observed in LipA and Est (Figure
4B) infiltrated rice leaves. In contrast to this, rice leaves co
infiltrated with LipA along with Agrobacterium strain EHA105
carrying pMDC7-xopQ (Figure 4D), pMDC7-xopX (Figure 4F)
or pMDC7-xopZ (Figure 4H) in the absence of Est exhibited
levels of callose deposits that were similar to those observed in
leaves infiltrated with LipA and Est (Figure 4B) (Figure 4I). As
observed for the suppression of LipA induced PCD in rice
roots, co-infiltration of LipA and EHA105/carrying Agrobacterial
T-DNA vector with any of the following genes xopF, xopK,
xopP, xopR, xopT, xopU, xopV, xopW, xopY, xopAA, xopAB or
avrBs2 did not result in suppression of callose deposition either
in the presence or absence of Est (data not shown). These
data indicated that xopX, xopQ or xopZ genes were able to
suppress LipA induced callose deposition in rice leaves.

A xopN- xopQ- xopX- xopZ- quadruple mutant of X.

oryzae pv. oryzae induces callose deposition in rice
leaves
A T3S deficient strain of X. oryzae pv. oryzae has been
shown to be an inducer of innate immune responses in rice
[22]. This has been attributed to its inability to suppress host
immune responses induced by DAMPs and possibly PAMPs.
The experiments described above indicate that the T3S
effectors XopN, XopQ, XopX and XopZ are involved in Figure 3. Agrobacterium mediated transient transfer of
suppression of LipA induced innate immunity in rice. This xopQ, xopX and xopZ genes of Xanthomonas oryzae pv.
prompted us to construct a xopN- xopQ- xopX- xopZ- quadruple oryzae suppresses LipA induced programmed cell death in
mutant of X. oryzae pv. oryzae and determine if it is able to, rice roots. Rice roots were treated with the following: buffer +
like a T3S - mutant, induce innate immunity in rice. The ability to Estradiol (Est; A); LipA + Est (B); pretreatment with EHA105/
induce callose deposition was assessed by infiltrating rice pMDC7-xopQ with Est (C); EHA105/pMDC7-xopX with Est (E)
leaves with wild type X. oryzae pv. oryzae, xopN- xopQ- xopX- and EHA105/pMDC7-xopZ with Est (G) followed by treatment
xopZ- quadruple mutant or T3S- mutant. Infiltration of wild type with LipA and Est (C, E and G); pretreatment with EHA105/
X. oryzae pv. oryzae (Figure 5B) resulted in induction of a pMDC7-xopQ (D); EHA105/pMDC7-xopX (F) and EHA105/
basal level of callose deposition that is no different from that pMDC7-xopZ (H) followed by treatment with LipA (D, F and H).
observed in water (Figure 5A) infiltrated leaves. Rice leaves The roots were subsequently stained with propidium iodide (PI)
infiltrated with xopN- xopQ- xopX- xopZ- quadruple mutant and visualized under a confocal microscope. Dispersed
(Figure 5C) exhibited higher amounts of callose deposits which intracellular PI staining is indicative of programmed cell death
were similar in amounts to those observed in leaves infiltrated in rice roots. Scale bar measures 20µm.
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 Suppression of Damage Induced Innate Immunity

Figure 4. Agrobacterium mediated transient transfer of xopQ, xopX and xopZ genes of Xanthomonas oryzae pv. oryzae
suppresses LipA induced callose deposition in rice leaves. Rice leaves were infiltrated with one of the following: buffer +
Estradiol (Est; A); LipA + Est (B); EHA105/pMDC7-xopQ + LipA with Est (C) or without Est (D); EHA105/pMDC7-xopX + LipA with
Est (E) or without Est (F); EHA105/pMDC7-xopZ + LipA with Est (G) or without Est (H). The leaves were subsequently stained with
aniline blue and visualized under an epifluorescence microscope. Callose deposition is seen as white spots in these pictures. Scale
bar measures 100µm. (I) Mean and standard deviation were calculated for number of callose deposits observed in an area of 0.60
mm2. Data were collected from at least five leaves per treatment in each experiment (three experiments indicated as ExpI, ExpII and
ExpIII) and two to three different viewing areas from the infiltrated region of each leaf. Statistically significant differences at P < 0.05
(Student’s two-tailed t test for independent means) were obtained from leaves co-infiltrated with LipA and EHA105 containing
pMDC7-xopQ, pMDC7-xopX or pMDC7-xopZ in the presence of Est as compared to the absence of Est.
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 Suppression of Damage Induced Innate Immunity

Figure 5. A xopN- xopQ- xopX- xopZ- quadruple mutant of Xanthomonas oryzae pv. oryzae induces callose deposition in
rice leaves. Rice leaves were infiltrated with one of the following: water (A); wild type Xoo (B); xopN- xopQ- xopX- xopZ- quadruple
mutant (C) and T3S- mutant (D). The treated leaves were subsequently stained with aniline blue and visualized under an
epifluorescence microscope. White dots in these pictures are indicative of callose deposition. Scale bar measures 100µm. (E) Mean
and standard deviation were calculated for number of callose deposits observed in a leaf area of 0.60 mm2. Data were collected
from at least five leaves per treatment in each experiment (three experiments indicated as ExpI, ExpII and ExpIII) and two to three
different viewing areas from the infiltrated region of each leaf. Statistically significant differences at P < 0.05 (Student’s two-tailed t
test for independent means) were obtained from leaves infiltrated with a xopN- xopQ- xopX- xopZ- quadruple mutant as compared to
leaves treated with either water or wild type. These differences were not observed in leaves infiltrated with xopN- xopQ- xopX- xopZ-
quadruple mutant as compared to leaves treated with T3S- mutant. WT Xoo = wild type X. oryzae pv. oryzae.
doi: 10.1371/[Link].0075867.g005

with the T3S- mutant (Figure 5D). In addition, like the T3S- xopQ- xopX- xopZ- quadruple mutant. Infiltration of a xopN-
mutant, the xopN- xopQ- xopX- xopZ- quadruple mutant induced xopQ- xopX- xopZ-/pHM1 (vector control) strain resulted in
PCD in rice roots (Figure S2). These data indicate that a X. callose amounts similar to those observed in xopN- xopQ-
oryzae pv. oryzae quadruple mutant that is defective in all four xopX- xopZ- infiltrated leaves (Figure S3). These data indicate
of the xopN, xopQ, xopX and xopZ genes induces innate that the enhanced ability of the quadruple mutant to induce
immune responses such as callose deposition and PCD to the immune responses is due to loss of an intact xopN gene and
same extent as a T3S- mutant strain of X. oryzae pv. oryzae. not due to a strange recombination event that might have
In order to assess the effect of addition of a single effector to occurred during creation of the xopN mutation in the triple
the quadruple mutant, we introduced the xopN gene into the mutant background during generation of the quadruple mutant.
xopN- xopQ- xopX- xopZ- quadruple mutant and assessed the Furthermore, the ability to induce callose deposition was
ability of the xopN complemented strain to induce callose assessed for X. oryzae pv. oryzae xopN-, xopQ-, xopX- or xopZ-
deposition in rice leaves. Rice leaves infiltrated with either single mutant strains. The number of callose deposits in rice
xopN- xopQ- xopX- xopZ-/xopN+ or xopQ- xopX- xopZ- triple leaves infiltrated with any single X. oryzae pv. oryzae effector
mutant exhibited similar amounts of callose deposits which mutant was comparable to those observed in wild type
were lower than those observed in leaves infiltrated with xopN- infiltrated leaves (Figure S4). Similarly, the number of callose

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 Suppression of Damage Induced Innate Immunity

deposits in rice leaves infiltrated with either one of two double oryzae pv. oryzae that were reported by Furutani and co-
mutants (xopN- xopX- and xopQ- xopX-) or the triple mutant workers [24] to be translocated into plant cells. In this study, we
(xopQ- xopX- xopZ-) were comparable to those observed in wild have demonstrated that Agrobacterium mediated transient
type infiltrated leaves (data not shown). These data expression of any one of the X. oryzae pv. oryzae genes,
demonstrate that multiple T3S effectors are required for full xopN, xopQ, xopX and xopZ, results in suppression of rice
suppression of rice immune responses. innate immune responses such as callose deposition and PCD
that are induced by treatment with the cell wall degrading
Mutations in xopN, xopQ, xopX and xopZ genes affect enzyme, LipA. For 12 other genes (namely xopF, xopK, xopP,
virulence of X. oryzae pv. oryzae on rice xopR, xopT, xopU, xopV, xopW, xopY, xopAA, xopAB and
A xopX- mutant of X. oryzae pv. oryzae was isolated by avrBs2-like protein) no evidence of suppressor activity was
creating an inframe deletion in the xopX gene of the wild type found under these assay conditions. It is possible that these
strain described in materials and methods. A xopN- xopX- proteins might function in suppression of innate immune
double mutant of X. oryzae pv. oryzae was created by responses induced by either PAMPs [28] or other kinds of
homologous plasmid integration of pK18mob-xopN in the xopX- DAMPs such as the AtPEP1 peptide [29]. At least for XopR this
background. The virulence of X. oryzae pv. oryzae strains appears to be true as Akimoto-Tomiyama et al. [14] have
xopN- mutant, xopN-/xopN+; which carries xopN for demonstrated that XopR is a suppressor of flagellin induced
complementation, xopN-/pHM1; vector control, xopX- mutant, innate immunity in Arabidopsis. The possibility that some of
xopX-/xopX+; which carries xopX for complementation, xopX-/ these twelve genes might encode functions that act together
pHM1; vector control and xopN- xopX- double mutant was (for example as a protein complex) in host cells to suppress
assessed by clip inoculation of rice leaves. Lesion lengths were innate immunity cannot be ruled out as our experimental
measured 7 days after inoculation (DAI). At 7 DAI, the xopN- methodology would not permit us to identify proteins that act in
mutant and xopX- mutant strains showed lesions significantly such a fashion.
reduced in length as compared to lesions produced by wild Jha and co-workers have demonstrated that a T3S- mutant of
type indicating a partial virulence deficiency. The partial X. oryzae pv. oryzae functions as an inducer of rice innate
virulence deficiency exhibited by xopN- mutant and xopX- immunity [22]. This appears to occur because this mutant is
mutants was complemented by the respective genes in trans unable to suppress immune responses induced by plant cell
as the lesions observed in rice leaves clip inoculated with wall degrading enzymes that it secretes and also because it
strains xopN-/xopN+ and xopX-/xopX+ were significantly longer produces some other DAMPs or PAMPs. We demonstrate in
than that seen in rice leaves inoculated with the xopN- mutant this paper that a xopN- xopQ- xopX- xopZ- quadruple mutant of
and xopX- mutant strains. The lesion lengths caused by the X. oryzae pv. oryzae induces callose deposition and PCD to
xopN- xopX- double mutant strain were significantly reduced the same extent as a T3S- mutant. This indicates that the xopN,
compared to the lesions produced by either xopN- mutant or xopQ, xopX and xopZ gene products play a major role in the
xopX- mutant strains (Figure 6A). This demonstrates that suppression of host innate immunity during infection. X. oryzae
mutations in xopN and xopX genes caused a partial virulence pv. oryzae strains having mutations in any one of the genes
deficiency that can be complemented by the expression of xopN, xopQ, xopX or xopZ retain the ability to suppress LipA
respective genes in trans and a xopN- xopX- double mutant induced innate immunity in rice. This indicates a functional
exhibited much more pronounced virulence deficiency than redundancy amongst these four gene products with regard to
either of the single mutants. their ability to suppress LipA induced immune responses.
The virulence proficiency of strains with single mutations in As the suppression of host defence responses is very crucial
the xopQ and xopZ genes was also assessed by inoculation of for the pathogen to cause disease, the T3Es that are
rice leaves. The results indicate that mutations in either xopQ suppressors of host innate immunity should have a role in
or xopZ cause a partial virulence deficiency on rice (Figure 6B). bacterial virulence on plants. We show that single mutations in
Introduction of complementing clones carrying xopQ or xopZ the xopN, xopQ, xopX or xopZ genes cause partial virulence
genes into their respective mutants leads to enhanced deficiency of X. oryzae pv. oryzae on rice. Song and Yang [13]
virulence on rice. have earlier shown that xopZ deficiency in X. oryzae pv. oryzae
strain PXO99A results in partial loss of virulence on rice. Taken
Discussion together, these results indicate that the xopN, xopQ, xopX and
xopZ genes are required for full virulence on rice. A xopN-
DAMPs released following plant cell wall degradation during xopX- double mutant exhibits an even greater virulence
pathogen attack serve as a mark of infection and induce innate deficiency than either one of the single mutants. This indicates
immune responses in the host. These DAMP induced host that these effectors may contribute independently in promoting
defense responses are suppressed by the pathogen during virulence on rice. The virulence deficiency associated with
infection. Jha et al. [22] showed the involvement of the type 3 each of the single mutations is in contrast to our observation
secretion system (T3S) of X. oryzae pv. oryzae in suppression that the single mutants are proficient at suppression of LipA
of cell wall damage induced innate immune responses in rice. induced innate immunity. This suggests that each of the four
We began our search for T3S effectors (T3Es) of X. oryzae pv. effector genes has a role in suppressing immune responses
oryzae that can suppress cell wall damage induced innate that are independent of those induced by LipA or cell wall
immune responses of rice by testing each of the 16 T3Es of X. damage. The possibility that each of these four effectors has

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 Suppression of Damage Induced Innate Immunity

Figure 6. Virulence deficiency associated with xopN-, xopQ-, xopX- or xopZ- single mutants of Xanthomonas oryzae pv.
oryzae. Leaves of susceptible rice variety Taichung-Native 1 (TN-1) were clip inoculated with the following X. oryzae pv. oryzae
strains: (A) wild type, xopN- mutant, xopN-/xopN+ (complemented strain), xopN-/pHM1 (vector control), xopX- mutant, xopX-/xopX+
(complemented strain), xopX-/pHM1 (vector control) or xopN- xopX- double mutant strains; (B) wild type, xopQ- mutant, xopQ-/xopQ+
(complemented strain), xopQ-/pHM1 (vector control), xopZ- mutant, xopZ-/xopZ+ (complemented strain) or xopZ-/pHM1 (vector
control). Lesion lengths were measured 7 days post inoculation. Error bars indicate the standard deviation of readings from at least
10 inoculated leaves. Similar results were obtained in independent experiments. A Student’s two-tailed t test for independent means
was performed for the following values: wild type with each of the single mutants and the double mutant with each of the single
mutants with correction for multiple comparisons, mutant with empty vector and complemented strains for each single mutant. The
brackets on the graphs indicate the comparisons that were made. All compared values are significantly different at P < 0.05 level.
doi: 10.1371/[Link].0075867.g006

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 Suppression of Damage Induced Innate Immunity

some other role in promoting virulence of X. oryzae pv. oryzae, [36] and X. campestris pv. armoraciae which has only XopR
other than in suppression of innate immunity, cannot also be and XopP [35]. The core effectors might be important for
ruled out. virulence and pathogen fitness in a wide range of host plants.
Why might X. oryzae pv. oryzae need multiple T3Es to Variable effectors are not uniformly distributed across
suppress cell wall damage induced innate immunity? The xanthomonads suggesting their possible role in specific host-
presence of a few functionally redundant proteins can be pathogen interactions. The observation that XopN, XopQ,
advantageous to the pathogen as it may ensure suppression of XopX and XopZ effectors are involved in suppression of cell
LipA/cell wall damage induced innate immunity even if any one wall damage induced innate immunity and the fact that they are
of the effector functions is for some reason compromised. Also, found in a wide variety of xanthomonads points to the
multiple effectors may ensure foolproof suppression of host possibility that suppression of cell wall damage induced innate
innate immunity by targeting the host signal transduction immunity is a key prerequisite for successful infection in a large
pathways at multiple steps. Our results indicate that XopN number of plants.
protein of X. oryzae pv. oryzae suppresses innate immune Ectopic expression of the xopZ gene of X. oryzae pv. oryzae
responses induced by treatment with LipA. We have also PXO99A strain suppressed defense-related callose deposition
shown that XopN suppresses innate immune responses induced by a T3S- mutant of X. oryzae pv. oryzae in a non-host
induced by CbsA (cellosiosidase), another cell wall degrading plant N. benthamiana [13]. X. oryzae pv. oryzae PXO99A strain
enzyme secreted by X. oryzae pv. oryzae (DIPANWITA SINHA has two identical paralogs of the xopZ gene [37] because of a
and RAMESH SONTI, unpublished results). This suggests that 212 kb duplication in the PXO99A genome that includes the
signaling pathways for DAMPs released by each of these cell xopZ gene. A double mutant in the xopZ1 and xopZ2 genes of
wall degrading enzymes might be converging in the cell. Kim X. oryzae pv. oryzae, and not either of the single mutants,
and co-workers showed that XopN protein of X. campestris pv. resulted in reduced lesion formation and growth in rice.
vesicatoria suppresses PTI in tomato [8]. The fact that XopN Introduction of either one of these two genes complemented
suppresses PTI and DTI suggests the possibility that certain the mutant phenotype and restored full virulence to the double
plant functions with which XopN can interact are common to mutant. In our study, we have shown that transient expression
signalling pathways induced by PAMPs and DAMPs. of X. oryzae pv. oryzae xopZ gene in rice results in
Specific biochemical activities have not yet been suppression of cell wall damage induced innate immunity. Our
demonstrated for XopN, XopX, XopZ and XopQ proteins. observation that a single mutation in the xopZ gene is sufficient
However, XopQ is predicted to have nucleoside hydolase (IU- to affect virulence indicates that the wild type X. oryzae pv.
NH) activity [7]. It has been speculated that the XopQ effector oryzae (the strain from India used in our study) carries a single
might be functioning to perturb signaling cascades that lead to copy of xopZ. In this respect, the wild type strain used in this
defence responses in the host by scavenging or sequestering a study is similar to X. oryzae pv. oryzae strain KACC 100331
nucleoside that may act as a messenger in signaling/metabolic from Korea [38] and X. oryzae pv. oryzae strain MAFF 311018
networks [7]. The XopN protein of X. campestris pv. vesicatoria from Japan [39] which have single copies of XopZ. Neither
shows structural homology to proteins containing tandemly Song and Yang [13] nor Zhao et al. [40] observed a reduction
repeated α-helices known as ARM/HEAT repeats [7] and found in symptoms for xopN, xopQ or xopX mutants of X. oryzae pv.
to interact with a 14-3-3 adapter protein of tomato [8,30]. The oryzae. We suggest that some other, as yet unidentified
ARM/HEAT repeats are typically found in eukaryotic proteins function, present in the genetic backgrounds of the wild type
that function in diverse cellular signaling networks such as strains used in those studies is redundant with these three
Arabidopsis homologs of nuclear transport protein importin-β effectors and that this function is not present in the genetic
[31] and AtCAND1 which functions in regulation of auxin background of the wild type strain used in our study.
signaling [32]. Furthermore, it has been shown that different In summary, we have found that four proteins of X. oryzae
isoforms of 14-3-3s interact with bacterial effector proteins in pv. oryzae namely XopN, XopQ, XopX and XopZ are
various plant-pathogen interactions to modulate plant innate suppressors of cell wall damage induced innate immunity in
immunity [30,33,34]. It is possible that the XopN protein of X. rice. This is the first report showing suppression of cell wall
oryzae pv. oryzae also interacts with rice 14-3-3s during damage induced innate immunity by bacterial T3Es in any
suppression of innate immunity. XopX is a methionine rich plant-pathogen interaction. The mechanisms by which these
protein whereas the XopZ protein is a conserved hypothetical proteins interfere with the elaboration of cell wall damage
protein without any known motifs. It is possible that XopX and induced rice innate immunity remains to be determined.
XopZ proteins do not have specific biochemical activities but
exert their effects by disrupting signalling pathways through Materials and Methods
interaction with specific host proteins.
The non-TAL effectors of xanthomonads have been further Bacterial strains, plasmids and growth conditions
categorized into core effectors and non-core/variable effectors The E. coli and X. oryzae pv. oryzae strains used in this
[35]. XopR, XopK, XopL, XopN, XopP, XopQ, XopX, XopZ and study are listed in Table 1. The Agrobacterial strains used in
AvrBs2 have been categorized as core effectors as they are this study are listed in Table 2. The plasmids used in this study
found to be conserved in all Xanthomonas spp. (whose are listed in Tables 3, 4 and 5. Oligonucleotide primers used in
genomes have been sequenced) with the exception of X. this study are listed in Tables S1 and S2. X. oryzae pv. oryzae
albilineans str. GPEPC73 which has none of these effectors strains were grown at 28°C in peptone sucrose (PS) medium,

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 Suppression of Damage Induced Innate Immunity

Table 4. Plasmids used in this study.

Plasmids Relevant characteristicsa Reference/Source

pUbi-C1300 with 624-bp fragment containing full length xopW gene from genomic DNA of wild type strain of Xoo cloned into KpnI and
pUbi-xopW This work
SacI site of pUbi-C1300; Kmr
pUbi-C1300 with 848-bp fragment containing full length xopY gene from genomic DNA of wild type strain of Xoo cloned into KpnI and
pUbi-xopY This work
SacI site of pUbi-C1300; Kmr
pUbi-C1300 with 964-bp fragment containing full length xopT gene from genomic DNA of wild type strain of Xoo cloned into KpnI and
pUbi-xopT This work
SacI site of pUbi-C1300; Kmr
pUbi-C1300 with 1746-bp fragment containing full length xopAA gene from genomic DNA of wild type strain of Xoo cloned into KpnI
pUbi-xopAA This work
and SacI site of pUbi-C1300; Kmr
pUbi-C1300 with 623-bp fragment containing full length xopAB gene from genomic DNA of wild type strain of Xoo cloned into KpnI
pUbi-xopAB This work
and SacI site of pUbi-C1300; Kmr
pUbi-C1300 with 2194-bp fragment containing full length xopP gene from genomic DNA of wild type strain of Xoo cloned into SmaI
pUbi-xopP This work
site of pUbi-C1300; Kmr
pENTR-avrBs2 pENTR/D/TOPO with 708-bp fragment containing full length avrBs2 gene from genomic DNA of wild type strain of Xoo; Kmr This work
pENTR-xopK pENTR/D/TOPO with 2538-bp fragment containing full length xopK gene from genomic DNA of wild type strain of Xoo; Kmr This work
pENTR-xopZ pENTR/D/TOPO with 3867-bp fragment containing full length xopZ gene from genomic DNA of wild type strain of Xoo; Kmr This work
pENTR-xopU pENTR/D/TOPO with 2958-bp fragment containing full length xopU gene from genomic DNA of wild type strain of Xoo; Kmr This work
pENTR-xopV pENTR/D/TOPO with 996-bp fragment containing full length xopV gene from genomic DNA of wild type strain of Xoo; Kmr This work
pENTR-xopX pENTR/D/TOPO with 2190-bp fragment containing full length xopX gene from genomic DNA of wild type strain of Xoo; Kmr This work
pENTR-xopR pENTR/D/TOPO with 1314-bp fragment containing full length xopR gene from genomic DNA of wild type strain of Xoo; Kmr This work
a Kmr and Spr indicate resistance to kanamycin and spectinomycin respectively. Xoo implies X. oryzae pv. oryzae.

doi: 10.1371/[Link].0075867.t004

Table 5. Plasmids used in this study.

Plasmids Relevant characteristicsa Reference/Source

pENTR-xopQ pENTR/D/TOPO with 1392-bp fragment containing full length xopQ gene from genomic DNA of wild type strain of Xoo; Kmr This work
pENTR-xopF pENTR/D/TOPO with 1986-bp fragment containing full length xopF gene from genomic DNA of wild type strain of Xoo; Kmr This work
pMDC7-avrBs2 pMDC7 with 708-bp fragment containing full length avrBs2 gene obtained from recombination of pENTR-avrBs2 with pMDC7; Spr This work
pMDC7-xopK pMDC7 with 2538-bp fragment containing full length xopK gene obtained from recombination of pENTR-xopK with pMDC7; Spr This work
pMDC7-xopV pMDC7 with 996-bp fragment containing full length xopV gene obtained from recombination of pENTR-xopV with pMDC7; Spr This work
pMDC7-xopZ pMDC7 with 3867-bp fragment containing full length xopZ gene obtained from recombination of pENTR-xopZ with pMDC7; Spr This work
pMDC7-xopX pMDC7 with 2190-bp fragment containing full length xopX gene obtained from recombination of pENTR-xopX with pMDC7; Spr This work
pMDC7-xopR pMDC7 with 1314-bp fragment containing full length xopR gene obtained from recombination of pENTR-xopR with pMDC7; Spr This work
pMDC7-xopQ pMDC7 with 1392-bp fragment containing full length xopQ gene obtained from recombination of pENTR-xopQ with pMDC7; Spr This work
pMDC7-xopU pMDC7 with 2958-bp fragment containing full length xopU gene obtained from recombination of pENTR-xopU with pMDC7; Spr This work
pMDC7-xopF pMDC7 with 1986-bp fragment containing full length xopF gene obtained from recombination of pENTR-xopF with pMDC7; Spr This work
a Kmr and Spr indicate resistance to kanamycin and spectinomycin respectively. Xoo implies X. oryzae pv. oryzae.

doi: 10.1371/[Link].0075867.t005

and E. coli strains were grown at 37°C in Luria Bertani (LB) method [43]. High-fidelity Phusion polymerase from Finnzymes
medium as described previously [41]. Agrobacterium (Thermo scientific, Rockford, IL, USA) was used for PCR
tumefaciens strains were grown in tryptone yeast extract amplification of insert DNA for all cloning purposes while Taq
mannitol (TYM) medium with appropriate antibiotics. The polymerase was used for all other applications. Restriction
concentrations of antibiotics used in this study were as follows: digestions were done with enzymes from New England Biolabs
rifampicin (Rf), 50 µg/ml; ampicillin (Ap), 100 µg/ml; (NEB, Beverly, MA, USA). PCR products and restriction
spectinomycin (Sp), 50 µg/ml; kanamycin (Km) 50 µg/ml for E. enzyme-digested DNA fragments were purified using a
coli and 15 µg/ml for X. oryzae pv. oryzae. QIAquick PCR purification kit and a QIAquick nucleotide
removal kit (Qiagen), respectively. Ligation with T4 DNA ligase,
Molecular biological techniques agarose gel electrophoresis, and transformation of E. coli were
performed as described previously [43].
Genomic DNA was isolated as described by Leach and co-
workers [42]. Plasmid DNA was isolated by using either a
Qiagen (Hilden, Germany) plasmid midi-kit or alkaline lysis

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 Suppression of Damage Induced Innate Immunity

Table 1. Bacterial strains used in this study. Table 2. Bacterial strains used in this study.

Reference/ A. tumefaciens Reference/

Strains Relevant characteristicsa Source Strains Relevant characteristicsa Source
E. coli DH5α C58, pTiBo542; T-region::aph, Kmr;
λ– f80dlacZDM15 D(lacZYA-argF)U169 recA1 EHA101 A281 derivative harbouring pEHA10, T- [53]
DH5α endA hsdR17 (rK– mK–) supE44 thi-1 gyrA Invitrogen DNA replaced with nptII, super-virulent
relA1 EHA105 Kms, derivative of EHA101 [54]
RP4-2 Tc::Mu-Km::Tn7 pro hsdR recA Tra+ EHA105 carrying plasmid pUbi-xopN ;
S17-1 [52] EHA105/ pUbi-xopN This work
used as a mobilizing strain Kmr
X. oryzae pv. oryzae EHA105 carrying plasmid pUbi-xopP ;
EHA105/ pUbi-xopP This work
BXO1 wild type; Indian isolate Laboratory* Kmr
Wild type rif-2; derivative of BXO1 Laboratory* EHA105 carrying plasmid pUbi-xopW ;
EHA105/ pUbi-xopW This work
xopN::pK18mob rif-2; Kmr; XopN-, derivative Kmr
xopN- This work
of wild type EHA105 carrying plasmid pUbi-xopY ;
EHA105/ pUbi-xopY This work
xopN::pK18mob rif-2/pDS2; xopN+, Kmr, Spr; Kmr
xopN-/xopN+ This work
derivative of xopN- EHA105 carrying plasmid pUbi-xopT ;
EHA105/ pUbi-xopT This work
xopN::pK18mob rif-2/pHM1; Kmr, Spr; Kmr
xopN-/pHM1 This work
derivative of xopN- EHA105/ pUbi- EHA105 carrying plasmid pUbi-xopAA ;
This work
∆xopX (243 aa-in-frame deletion) rif-2; XopX-, xopAA Kmr
xopX- This work
derivative of wild type EHA105/ pUbi- EHA105 carrying plasmid pUbi-xopAB ;
This work
∆xopX rif-2/pDS4; xopX+, Kmr, Spr; derivative xopAB Kmr
xopX-/xopX+ This work
of xopX- EHA105 carrying plasmid pUbi (vector
EHA105/ pUbi This work
∆xopX rif-2/pHM1; Kmr, Spr; derivative of control); Kmr
xopX-/pHM1 This work
xopX- EHA105/ pMDC7- EHA105 carrying plasmid pMDC7-
This work
∆xopQ (171 aa-in-frame deletion) rif-2; xopR xopR; Spr
xopQ- This work
XopQ-, derivative of wild type EHA105/ pMDC7- EHA105 carrying plasmid pMDC7-
This work
xopQ-/xopQ+ ∆xopQ rif-2/pDS6 XopQ+, derivative of xopQ- This work xopV xopV; Spr
xopQ-/pHM1 ∆xopQ rif-2/pHM1; XopQ-, derivative of xopQ- This work EHA105/ pMDC7- EHA105 carrying plasmid pMDC7-
This work
∆xopZ (431 aa-in-frame deletion) rif-2; XopZ-, xopK xopK; Spr
xopZ- This work
derivative of wild type EHA105/ pMDC7- EHA105 carrying plasmid pMDC7-
This work
xopZ-/xopZ+ ∆xopZ rif-2/pDS8; XopZ+, derivative of xopZ- This work xopF xopF; Spr
xopZ-/pHM1 ∆xopZ rif-2/pHM1; XopZ-, derivative of xopZ- This work EHA105/ pMDC7- EHA105 carrying plasmid pMDC7-
This work
xopN::pK18mob rif-2; Kmr; XopN- XopX-; xopQ xopQ; Spr
xopN- xopX- This work
derivative of xopX- EHA105/ pMDC7- EHA105 carrying plasmid pMDC7-
This work
xopQ- xopX- ∆xopQ rif-2; XopQ- XopX-, derivative of xopX- This work xopU xopU; Spr
xopQ- xopX- ∆xopZ rif-2; XopQ-XopX- XopZ-, derivative of EHA105/ pMDC7- EHA105 carrying plasmid pMDC7-
This work This work
xopZ- xopQ- xopX- xopX xopX; Spr
xopN- xopQ- xopN::pK18mob rif-2; Kmr; XopN- XopQ- EHA105/ pMDC7- EHA105 carrying plasmid pMDC7-
This work This work
xopX- xopZ- XopX- XopZ-derivative of xopQ- xopX- xopZ- xopZ xopZ; Spr
xopN- xopQ- xopN- xopQ- xopX- xopZ- rif-2/pDS2; XopN+, EHA105/ pMDC7- EHA105 carrying plasmid pMDC7-
This work
xopX- xopZ-/ Kmr, Spr; derivative of xopN- xopQ-xopX- This work avrBs2 avrBs2; Spr
xopN+ xopZ- a Kms indicates sensitivity to kanamycin. Kmr and Spr indicate resistance to

xopN-xopQ- kanamycin and spectinomycin

xopN- xopQ- xopX- xopZ- rif-2/pHM1; Kmr,
xopX- xopZ-/ This work respectively.
Spr; derivative of xopN- xopQ- xopX- xopZ-
pHM1 doi: 10.1371/[Link].0075867.t002
hrpB6: :bla rif-2; T3S- Hrp-, Apr derivative of
T3S- [22]
wild type
USA). BLAST algorithm in the NCBI database [44] was used
a The rif-2 mutation confers resistance to rifampicin; Apr, Kmr and Spr indicate
for homology and conserved domain searches. No new DNA
resistance to ampicillin, kanamycin and spectinomycin respectively. * Laboratory
sequence has been generated in this study.
collection
doi: 10.1371/[Link].0075867.t001
Construction and complementation of xopN-, xopQ-,
xopX- and xopZ-, mutants of X. oryzae pv. oryzae
DNA sequencing and bioinformatics tools
A xopN- mutant of X. oryzae pv. oryzae was generated by
DNA sequencing was done with an ABI Prism 3700
gene disruption using homologous plasmid integration of a
automated DNA sequencer (PerkinElmer, Foster City, CA,
suicide vector pK18mob [45]. A 497-bp internal fragment of the

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 Suppression of Damage Induced Innate Immunity

Table 3. Plasmids used in this study. fragment was cloned into the SmaI digested blunt-ended
pK18mob cloning vector to obtain recombinant plasmid pDS1.
The plasmid pDS1 was then electroporated into wild type and
Reference/ clones were selected for kanamycin resistance to obtain a
Plasmids Relevant characteristicsa Source xopN- mutant by plasmid integration. Gene disruption was
pUC18 derivative; Mob+ Tra- Kmr; does not confirmed by PCR using xopN gene-specific flanking primers
pK18mob [45] XopNF/XopNR in combination with vector-specific primers
replicate in Xoo
pK18mobSacB derivative of pK18mob, sucrose sensitive [45] M13F and M13R and sequencing of PCR products. The xopN
pHM1 Broad host range cosmid vector (~13.3 kb); Spr [48] gene fragment was cloned in the same transcriptional
PUbi pUbi-C1300 binary cloning vector; Kmr [26] orientation as the lacZ promoter of pK18mob vector in pDS1 so
17-β-estradiol inducible binary cloning vector; that mutation caused by pDS1 integration does not have a
pMDC7 [51]
Spr polar effect on downstream genes due to activity of the
pENTR/D/TOPO cloning vector; Kmr Invitrogen outwardly directed lacZ promoter [46,47].
pK18mob with 497-bp internal fragment of xopN For xopN- mutant complementation, a 2225-bp DNA
pDS1 gene of wild type strain of Xoo cloned into SmaI This work fragment containing the full length of xopN gene was amplified
site of pk18mob by PCR using primers XopN2F/XopN2R and total genomic
pHM1 with 2225-bp fragment containing full DNA of X. oryzae pv. oryzae as template. The amplified DNA
length xopN gene from genomic DNA of wild fragment was cloned as a HindIII-SacI fragment into the broad
pDS2 This work
type strain of Xoo cloned into HindIII and SacI host range vector pHM1 [48] to create the recombinant plasmid
sites of pHM1; Spr pDS2. The pDS2 construct and the pHM1 vector were
pK18mobSacB with 1473-bp fragment of xopX individually introduced into the xopN-mutant strain by
gene of wild type strain of Xoo with an inframe electroporation and selection for spectinomycin resistance. The
pDS3 internal deletion of 729-bp (243aa) and This work presence of pDS2 was confirmed by PCR using xopN gene-
insertion of 12 bp XbaI site cloned into HindIII specific flanking primers XopN2F/XopN2R and universal
site of pk18mobSacB primers M13F/M13R and sequencing of PCR products. The
pHM1 with 2190-bp fragment containing full confirmed clones were designated as xopN-/xopN+ (xopN-/
length xopX gene from genomic DNA of wild pDS2; for complementation) and xopN-/pHM1 (xopN-/pHM1;
pDS4 This work
type strain of Xoo cloned into HindIII and SacI vector control) and studied further.
sites of pHM1; Spr A xopX- mutant of X. oryzae pv. oryzae was isolated by
pK18mobSacB with 882-bp fragment of xopQ creating an inframe deletion in the xopX gene. For this
gene of wild type strain of Xoo with an inframe purpose, 729 and 732 bp fragments of the xopX gene were
pDS5 internal deletion of 513-bp (171aa) and This work amplified by PCR using genomic DNA of wild type strain and
insertion of 12 bp XbaI site cloned into HindIII the gene-specific XopXS1F/XopXS1R and XopXS3F/XopXS3R
site of pk18mobSacB; Kmr primer pairs, respectively. The primers XopXS1F and
pHM1 with 1422-bp fragment containing full XopXS3R were having an engineered site for restriction
length xopQ gene from genomic DNA of wild enzyme HindIII at their 5’ and 3’ ends, respectively. Similarly,
pDS6 This work
type strain of Xoo cloned into HindIII and SacI the primers XopXS1R and XopXS3F were having an
sites of pHM1; Spr engineered site for restriction enzyme XbaI at their 3’ and 5’
pK18mobSacB with 2570-bp fragment of xopZ ends, respectively. The amplified fragments were digested with
gene of wild type strain of Xoo with an inframe XbaI restriction enzyme. The XbaI-digested 729 and 732 bp
pDS7 internal deletion of 1293-bp (431aa) and This work fragments were ligated and the ligated product was PCR
insertion of 12 bp XbaI site cloned into HindIII amplified using the gene-specific XopXS1F/XopXS3R primer
site of pk18mobSacB pair. A 1473 bp fragment was obtained which included a 12 bp
pHM1 with 3894-bp fragment containing full
insertion that contains the XbaI site. The 1461bp fragment,
length xopZ gene from genomic DNA of wild
pDS8 This work which now contained HindIII sites at the 5’ and 3’ ends, was
type strain of Xoo cloned into HindIII and SacI
digested with HindIII restriction enzyme and cloned into the
sites of pHM1; Spr
HindIII site of the pK18mobsacB cloning vector to obtain the
pUbi-C1300 with 2193-bp fragment containing
recombinant plasmid pDS3. Sequencing of pDS3 confirmed
full length xopN gene from genomic DNA of wild
pUbi-xopN This work that no other changes had occurred in the target xopX
type strain of Xoo cloned into SmaI site of pUbi-
sequence except an inframe deletion of 729 bp (243 aa) and
C1300; Kmr
insertion of 12 bp (XbaI restriction enzyme site along with
a Kmr and Spr indicate resistance to kanamycin and spectinomycin respectively.
adapter sequences). This construct was then transferred from
Xoo implies X. oryzae pv. oryzae.
E. coli S17-1 to wild type via biparental mating. The
doi: 10.1371/[Link].0075867.t003
transformants were plated on nutrient agar (Difco, Detroit, MI,
USA) plates containing Km. Single recombinant colonies that
xopN gene was amplified by PCR using genomic DNA of X. were kanamycin-resistant and sucrose-sensitive were grown
oryzae pv. oryzae wild type strain and the gene-specific and passaged thrice in nutrient broth without Km and sucrose.
XopN1F/XopN1R (Table S1) oligonucleotide primer pair. This Saturated bacterial cultures were dilution plated on PSA plates

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 Suppression of Damage Induced Innate Immunity

containing 1% sucrose to obtain double recombinant strains mutants. Briefly, the full length copies of xopQ and xopZ genes
that were sucrose-resistant and kanamycin-sensitive. The were PCR amplified from X. oryzae pv. oryzae genomic DNA
inframe deletion that had been incorporated into the xopX gene using primer pairs, XopQ2F/XopQ2R and XopZ2F/XopZ2R,
on the X. oryzae pv. oryzae chromosome was confirmed by respectively. PCR fragments of xopQ and xopZ genes were
PCR using xopX gene-specific flanking primers XopXS1F/ individually cloned as HindIII-SacI fragment in vector pHM1 to
XopXS3R and sequencing of PCR products. One of the create the constructs pDS6 and pDS8, respectively. The pDS6
confirmed mutant clones was designated xopX- mutant and and pDS8 constructs were individually introduced by
chosen for further study. Using a similar strategy, xopQ- and electroporation into the xopQ- mutant and xopZ- mutants,
xopZ- mutants of X. oryzae pv. oryzae were isolated by creating respectively, to get spectinomycin resistant recombinant
inframe deletions in the xopQ and xopZ genes. Briefly, 420 and clones. The presence of pDS6 and pDS8 was confirmed by
450 bp fragments of the xopQ gene were PCR amplified using PCR using respective gene-specific flanking primers XopQ2F/
genomic DNA of wild type strain and the gene-specific XopQ2R and XopZ2F/XopZ2R, and universal primers M13F/
XopQS1F/XopQS1R and XopQS3F/XopQS3R primer pairs, M13R and sequencing of PCR products. The confirmed clones
respectively. These PCR amplified fragments were then ligated for complementation of xopQ- and xopZ- mutants were
as XbaI-digested fragments. Similarly, 1304 and 1254 bp PCR designated as xopQ-/xopQ+ (xopQ-/pDS6) and xopZ-/xopZ+
fragments of the xopZ gene were obtained using genomic DNA (xopZ-/pDS8) respectively. For vector control, pHM1 vector was
of wild type strain and the gene specific XopZS1F/XopZS1R individually introduced by electroporation into the xopQ- mutant
and XopZS3F/XopZS3R primer pairs, respectively. These PCR and the xopZ- mutant. Spectinomycin resistant clones were
amplified fragments were digested with XbaI and ligated to designated as xopQ- /pHM1 (vector control) and xopZ- /pHM1
each other. The corresponding ligated products of xopQ and (vector control) and used for further study.
xopZ genes were PCR amplified as 882 and 2570 bp
fragments which included a 12 bp insertion that contains the Construction of xopN- xopX- double and xopN- xopQ-
XbaI site, using XopQS1F/XopQS3R and XopZS1F/XopZS3R xopX- xopZ- quadruple mutants of X. oryzae pv. oryzae
primer pairs, respectively. These fragments were digested with A xopN- xopX- double mutant of X. oryzae pv. oryzae was
HindIII and cloned individually into the HindIII site of isolated by gene disruption of xopN using homologous plasmid
pK18mobsacB to obtain the recombinant plasmids pDS5 and integration in a xopX- mutant strain. The construct pDS1 was
pDS7, respectively. The constructs pDS5 and pDS7 were electroporated into electrocompetent cells of xopX- mutant
confirmed to carry no other changes in the target xopQ and strain to obtain a xopN- xopX- double mutant by plasmid
xopZ gene sequences except inframe deletions of 513 bp (171 integration. The xopN gene disruption in xopN- xopX- mutant
aa) and 1293 bp (431 aa), respectively, and insertions of 12 bp strain was confirmed as described above for the mutation in
(XbaI restriction enzyme site along with adapter sequences). xopN- strain.
These constructs were then transferred from E. coli S17-1 to A xopQ- xopX- double mutant of X. oryzae pv. oryzae was
wild type via biparental mating and marker free xopQ- and isolated by creating an inframe deletion of 513 bp (171 aa) in
xopZ- mutants were generated following the steps as described the xopQ gene in the genome of xopX- mutant strain. For this
above for generation of xopX- mutant strain. The inframe purpose, the construct pDS5 was transferred from E. coli S17-1
deletions that had been incorporated into the xopQ and xopZ to xopX- mutant via biparental mating. An inframe deletion was
genes on the X. oryzae pv. oryzae chromosome were created in the xopQ gene in the genetic background of the
confirmed by PCR using xopQ and xopZ gene-specific flanking xopX- mutant. One of the confirmed clones was designated as
primers XopQS1F/XopQS3R and XopZS1F/XopZS3R xopQ- xopX- double mutant strain.
respectively, and sequencing of PCR products. The mutants for In a similar way, a xopQ- xopX- xopZ- triple mutant of X.
xopQ and xopZ genes were designated as xopQ- and xopZ-, oryzae pv. oryzae was generated by incorporating an inframe
respectively. deletion of 1293 bp (431 aa) in the xopZ gene in the genome of
For xopX- mutant complementation, a 2190 bp DNA fragment xopQ- xopX- double mutant strain. To achieve this, biparental
containing the full length of xopX gene was amplified by PCR mating was set up to mobilize the construct pDS7 from E. coli
using primers XopX2F/XopX2R and total genomic DNA of X. S17-1 to xopQ- xopX- double mutant. The inframe deletion that
oryzae pv. oryzae as template. The amplified DNA fragment had been incorporated in the xopZ gene in the genomic
was cloned as a HindIII-SacI fragment into the broad host background of xopQ- xopX- double mutant strain was confirmed
range vector pHM1 [48] to create the recombinant plasmid as described earlier for isolating the xopZ- mutant strain. One of
pDS4. The pDS4 plasmid and pHM1 vector were individually the confirmed clones was designated as a xopQ- xopX- xopZ-
introduced into the xopX- mutant strain by electroporation and triple mutant of X. oryzae pv. oryzae. A xopN- xopQ- xopX-
the recombinants were isolated by selection for spectinomycin xopZ- quadruple mutant of X. oryzae pv. oryzae was isolated by
resistance. The presence of pDS4 was confirmed by PCR disrupting the xopN gene using homologous plasmid
using xopX gene-specific flanking primers XopX2F/XopX2R integration in the xopZ- xopQ- xopX- triple mutant strain. A
and universal primers M13F/M13R and sequencing of PCR disruption of the xopN gene was achieved by electroporating
products. The confirmed clones were designated as xopX-/ the construct pDS1 into xopQ- xopX- xopZ- triple mutant strain
xopX+ (xopX-/pDS4; for complementation) and xopX-/pHM1 and selecting for kanamycin resistant transformants.
(xopX-/pHM1; vector control) and studied further. A similar Generation of the xopN- mutant was confirmed by PCR using
strategy was used for complementation of xopQ- and xopZ- xopN gene-specific flanking primers XopNF/XopNR in

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 Suppression of Damage Induced Innate Immunity

combination with vector-specific primers M13F and M13R and from saturated cultures of Agrobacterium strains, washed and
sequencing the PCR products. One of the confirmed clones resuspended in induction medium (10 mM MES, pH 5.6, 10
was designated as xopN- xopQ- xopX- xopZ- quadruple mutant mM MgCl2 and 150 µM acetosyringone) and then incubated in
of X. oryzae pv. oryzae. For complementation of xopN- xopQ- a shaker for 2 h at 28°C before infiltration [8]. Agrobacterium
xopX- xopZ- quadruple mutant with xopN gene, the pDS2 strains were recollected from induction medium by
plasmid was introduced into the xopN- xopQ- xopX- xopZ- centrifugation and processed further. Rice leaves were
quadruple mutant by electroporation and selection of coinfiltrated with Agrobacterium strains resuspended in buffer
spectinomycin resistant colonies. The presence of pDS2 was containing purified LipA (~100 µg/ml). Agrobacterium strains
confirmed by PCR. As a control, the pHM1 vector was containing the pMDC7 vector were resuspended in purified
introduced into the xopN- xopQ- xopX- xopZ- quadruple mutant LipA (~100 µg/ml) with and without 17-β-estradiol (20 µM final
in a similar manner. concentration). All the bacterial strains (X. oryzae pv. oryzae
and Agrobacterium) were used to final concentration of
Cell death assay OD=1.0. After 20-24 h, the infiltrated zones (~1× 1 cm) were
Surface sterilized rice (Taichung Native-1; a susceptible rice cut from the leaves, destained with 70% ethanol at 65°C,
variety) seeds were germinated on 0.5% sterile agar in Petri stained with 0.5% aniline blue for 4 h and observed under an
dishes for 2 to 3 days. Root tips, 1 to 2 cm long, were excised Axioplan2 epifluorescence microscope (Carl Zeiss, Germany)
from the seedlings and treated with 200 µl of either purified using a blue filter (excitation wavelength of 365 nm) and ×10
LipA (500 µg/ml) or buffer (10 mM phosphate buffer, pH 6.0). objective [49].
After incubation for 18-20 h, roots were washed and stained
with propidium iodide (PI) for 20 min and mounted in 50% Virulence assay
glycerol on glass slides. The samples were viewed under a For virulence analysis various X. oryzae pv. oryzae strains
LSM-510 Meta confocal microscope (Carl Zeiss, Germany) were grown to saturation in PS broth supplemented with
using 63 × oil immersion objectives and He-Ne laser at 543 nm appropriate antibiotics. The cells were pelleted down by
excitation to detect PI internalization. Longitudinal optical centrifugation, washed and resuspended in sterile water at a
sections of 1.0 µm thickness were acquired and further concentration of OD=1.0. Surgical scissors dipped in these
projected to obtain the image of 2 to 3 µm total thickness. All bacterial suspensions were used to clip the leaf tips of
images were analyzed using LSM software and further edited greenhouse-grown, 40-day-old plants of the susceptible rice
using Photoshop (Adobe, San Jose, CA, USA). variety Taichung Native-1 (TN-1) [50]. Lesion lengths were
measured 7 days after inoculation.
Preinoculation with Agrobacterium tumefaciens
EHA105 strains or X. oryzae pv. oryzae strains Cloning of T3S effectors of X. oryzae pv. oryzae into
X. oryzae pv. oryzae strains were grown in PS broth to Agrobacterial T-DNA vectors
saturation, pelleted, washed with buffer (10 mM phosphate Full length xopN and xopP genes were amplified by PCR
buffer, pH 6.0) and resuspended in buffer. The cells from using genomic DNA of wild type strain of X. oryzae pv. oryzae
saturated cultures of Agrobacterium strains grown in TYM broth and the corresponding gene-specific oligoneucletide primer
were pelleted, washed following centrifugation and pair (Table S1) and cloned into the SmaI site of the binary
resuspended in induction medium (10 mM MES, pH 5.6, 10 vector pUbi-C1300 [26] between the maize ubiquitin promoter
mM MgCl2, and 150 µM acetosyringone; Sigma) and then and nos3’ terminator to obtain recombinant plasmids pUbi-
incubated in a shaker for 2 h at 28°C [8]. Rice roots were xopN and pUbi-xopP respectively. Full length xopW, xopY,
pretreated with either suspensions of Agrobacterium strains for xopT, xopAA and xopAB genes were PCR amplified using
4-5 h in induction media, in the presence or absence of 17-β- genomic DNA of wild type X. oryzae pv. oryzae strain and the
estradiol (20 µM final concentration), or X. oryzae pv. oryzae corresponding gene-specific oligonucleotide primer pairs
strains dissolved in phosphate buffer for 2 h at 28°C. After (Table S1). The PCR products were cloned individually as
rinsing with fresh buffer, the roots were treated with purified KpnI-SacI fragments into the binary vector pUbi-C1300 to
LipA (~500 µg/ml) for 18-20 h at 28°C and processed further obtain the recombinant plasmids pUbi-xopW, pUbi-xopY, pUbi-
for confocal analyses as described above. As controls, rice xopT, pUbi-xopAA and pUbi-xopAB respectively. Positive
roots pretreated with either Agrobacterium strains or X. oryzae clones were screened using gene-specific and vector-specific
pv. oryzae strains and subsequently treated with buffer for primers and confirmed by sequencing. The full length genes of
18-20 h at 28°C were also analysed by confocal microscopy. xopR, xopV, xopK, xopF, xopQ, xopU, xopX, xopZ and avrBs2
All the bacterial strains were used at a final concentration of were PCR amplified from genomic DNA of wild type X. oryzae
OD=1.5. At least 200 µl volumes of either of LipA, buffer or pv. oryzae strain with engineered 5’ CACC overhang were
culture were used to treat the rice roots. cloned individually into cloning vector pENTR/D/TOPO
(Invitrogen, Carlsbad, CA, USA) to obtain plasmids pENTR-
Callose deposition assay xopR, pENTR-xopV, pENTR-xopK, pENTR-xopF, pENTR-
Leaves of 10-day old rice seedlings were infiltrated using the xopQ, pENTR-xopU, pENTR-xopX, pENTR-xopZ and pENTR-
blunt end of a 1 ml syringe with one of the following : buffer, avrBs2 respectively. Positive clones in E. coli DH5α were
water, purified LipA (~100 µg/ml) and saturated culture of X. screened using gene-specific primers and confirmed by
oryzae pv. oryzae T3S- mutant strain. Bacteria were collected sequencing. Plasmids from these clones were isolated and

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 Suppression of Damage Induced Innate Immunity

recombined into the 17-β-estradiol inducible binary cloning pMDC7-xopQ with Estradiol (Est) or without Est; EHA105/
vector pMDC7 [51] by Gateway LR reaction (Invitrogen) to pMDC7-xopR with Est or without Est; EHA105/pMDC7-xopX
obtain recombinant plasmids pMDC7-xopR, pMDC7-xopV, with Est or without Est; EHA105/pMDC7-xopZ with Est or
pMDC7-xopK, pMDC7-xopF, pMDC7-xopQ, pMDC7-xopU, without Est. Subsequently, each cDNA was used as a template
pMDC7-xopX, pMDC7-xopZ and pMDC7-avrBs2 respectively. for PCR amplification with Taq polymerase using gene-specific
Clones obtained in E. coli DH5α were screened using PCR with primers (Table S2) designed to amplify a 118-164 bp fragment
appropriate primer pairs and confirmed by sequencing. of each gene of interest. (A) PCR products of 123 bp
Plasmids containing cloned genes in the binary vectors (either (XopNRTF/XopNRTR primer pair) and 164 bp (XopPRTF/
pUbi-C1300 or pMDC7) were isolated and further XopPRTR primer pair) were obtained using, as a template,
electroporated into electrocompetent cells of A. tumefaciens cDNA prepared with RNA from roots treated with EHA105/
strain EHA105. pUbi-xopN and EHA105/pUbi-xopP, respectively. These
products were not obtained when cDNA prepared from roots
RT-PCR analysis to determine expression of xop genes treated with EHA105/pUbi was used as a template. PCR
of X. oryzae pv. oryzae in rice roots product of 158 bp (GAPDHF/GAPDHR primer pair which
Rice roots were treated with Agrobacterium EHA105 strains amplifies a fragment from the rice GAPDH gene) was obtained
carrying any one of xopN, xopP, xopQ, xopR, xopX or xopZ using cDNA prepared from roots treated with any of the three
genes on the T-DNA vector. The xopQ, xopR, xopX and xopZ Agrobacterial strains. (B) PCR products of 148 bp (XopQRTF/
genes are cloned in a T-DNA vector, pMDC7, wherein the XopQRTF primer pair), 128 bp (XopRRTF/XopRRTF primer
expression of the cloned gene is under the induction of 17-β- pair), 143 bp (XopXRTF/XopXRTF primer pair) and 149 bp
Estradiol (Est). The xopN and xopP is cloned in T-DNA vector (XopZRTF/XopZRTF primer pair) were obtained using, as a
pUbi-C1300, wherein its expression is under the control of template, cDNA prepared with RNA from roots treated with
maize ubiquitin promoter. Accordingly, as negative controls, for EHA105/pMDC7-xopQ, EHA105/pMDC7-xopR, EHA105/
the genes cloned in Est inducible vector (pMDC7), rice roots pMDC7-xopX and EHA105/pMDC7-xopZ in the presence of
were treated with Agrobacterial strains in the absence of Est, Est but not in the absence of Est, respectively. A PCR product
whereas for genes cloned in vector pUbi-C1300, rice roots of 158 bp (GAPDHF/GAPDHR primer pair) was obtained using,
were treated with Agrobacterium strain containing an empty as a template, cDNA obtained from roots treated with any of
vector. Total RNA was isolated from rice tissue treated with the above vectors either in the presence or absence of
different Agrobacterium EHA105 strains (with or without Est estradiol.
wherever needed) using Trizol reagent (Invitrogen) according (TIF)
to the manufacturer’s instructions. The quality of isolated RNA
was assessed by agarose gel electrophoresis and quantitated Figure S2. A xopN- xopQ- xopX- xopZ- quadruple mutant of
using spectrophotometer (Nanodrop ND-1000, Thermo Xanthomonas oryzae pv. oryzae induces PCD in rice roots.
Scientific). 5 µg of total RNA samples were treated with Rice roots were treated with one of the following: wild type X.
RNasefree DNaseI (Promega) for 1 h at 37°C to remove any oryzae pv. oryzae (A); xopN- xopQ- xopX- xopZ- quadruple
DNA contamination. After inactivation of DNaseI, 1 µg RNA mutant (B); T3S- mutant (C). Treated roots were subsequently
was used for cDNA synthesis using SuperscriptIII (Invitrogen) stained with propidium iodide (PI) and viewed under a confocal
RT enzyme as per the manufacturer’s instructions and oligo microscope. Internalisation of PI is indicative of defense
(dT) primers. Subsequently, each cDNA was used as template response-associated programmed cell death in rice roots.
for PCR amplification with Taq polymerase using gene-specific Scale bar measures 20µm.
primers (Table S2) designed to amplify fragments of each gene (TIF)
of interest. The DNaseI treated RNA that was not reverse
transcribed was used as template in PCR as negative control Figure S3. Complementation with the xopN gene reduces
to show that the RT-PCR products obtained were not because ability of the xopN- xopZ- xopQ- xopX- quadruple mutant to
of any DNA contamination in the cDNA samples. The induce callose deposition in rice leaves. Rice leaves were
amplification condition used was 95°C for 5 min followed by 28 infiltrated with one of the following: xopQ- xopX- xopZ- triple
cycles of 94°C for 30 sec, 58°C for 30 sec, 72°C for 45 sec, mutant, xopN- xopQ- xopX- xopZ- quadruple mutant, xopN-
again followed by 72°C for 10 min. The RT-PCR amplified xopQ- xopX- xopZ- /xopN+ (quadruple mutant complemented
products were analysed by agarose gel electrophoresis and with xopN gene) and xopN- xopQ- xopX- xopZ-/pHM1
staining with ethidium bromide. (quadruple mutant with pHM1 plasmid; vector control). The
leaves were subsequently stained with aniline blue and
Supporting Information visualized under an epifluorescence microscope. Callose
deposits were quantified from 0.60 mm2 area of an infiltrated
Figure S1. Expression of xopN, xopP xopQ, xopR, xopX leaf. Data were collected from atleast five leaves in each
and xopZ genes of Xanthomonas oryzae pv. oryzae in rice experiment (three experiments indicated as ExpI, ExpII and
roots following Agrobacterium mediated transient transfer. ExpIII) and 2-3 different viewing areas from the infiltrated
cDNA was synthesised from total RNA isolated from rice roots region of each leaf. Statistically significant differences at P <
treated with one of the following: EHA105/pUbi-xopN; 0.05 (Student’s two-tailed t test for independent means) were
EHA105/pUbi-xopP; EHA105/pUbi (empty vector); EHA105/ obtained from leaves infiltrated with either xopN- xopQ- xopX-

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 Suppression of Damage Induced Innate Immunity

xopZ-/xopN+ or xopQ- xopX- xopZ- triple mutant as compared to (DOCX)

leaves treated with a xopN- xopQ- xopX- xopZ- quadruple
mutant. Statistically significant differences were not observed in Table S2. List of oligonucleotide primers used for RT-
the following comparisons: either xopN- xopQ- xopX- xopZ-/ PCR.
xopN+ with xopQ- xopX- xopZ- triple mutant or xopN- xopQ- (DOCX)
xopX- xopZ- with xopN- xopZ- xopQ- xopX-/pHM1.
(TIF) Acknowledgements
Figure S4. xopN-, xopQ-, xopX- or xopZ- single mutants of We thank Max Dow for providing the plasmid pK18mobsacB.
Xanthomonas oryzae pv. oryzae induce callose deposition We thank Nam Hai Chua for providing the plasmid pMDC7. We
at a basal level. Rice leaves were infiltrated with one of the thank Pamela Ronald and R. Rajeshwari for providing the
following: wild type X. oryzae pv. oryzae, xopN- mutant, xopQ- plasmid pUbi-C1300. We acknowledge the help of C.
mutant, xopX- mutant, xopZ- mutant. The leaves were Subbalakshmi and V. Krishna Kumari for help in enzyme
subsequently stained with aniline blue and visualized under an purification, and R. Nandini for confocal analysis.
epifluorescence microscope. Callose deposits were quantified
from 0.60 mm2 area of an infiltrated leaf. Data were collected Author Contributions
from atleast five leaves in each experiment and 2-3 different
viewing areas from the infiltrated region of each leaf. Data from Conceived and designed the experiments: DS RVS. Performed
one experiment are represented. Similar results were obtained the experiments: DS MKG HKP AR. Analyzed the data: DS
in independent experiments. RVS. Contributed reagents/materials/analysis tools: DS. Wrote
(TIF) the manuscript: DS RVS.

Table S1. List of oligonucleotide primers used in this

study.

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