Mol. Cells 2016; 39(5): 426-438
Published online May 3, 2016
https://doi.org/10.14348/molcells.2016.0094
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: nsjwa@sejong.ac.kr
Plant disease resistance occurs as a hypersensitive response (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant immune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the
Keywords AVR-effectors, gene-for-gene interaction, NADP-Malic enzyme, reactive oxygen species, rice
Many scientists have striven to find ways to control hemibiotrophic
Pathogen AVR effectors have been genetically proven to be essential components in plant immune responses (Flor, 1971). However, the mechanism by which AVR effectors and R proteins are associated with these responses remains unclear. Initially, the ligand-receptor model (Gabriel and Rolfe, 1990) was widely supported, but the lack of physical interactions between a number of R/AVR pairs has resulted in the generation of alternative guard and decoy hypothesis (McHale et al., 2006; van der Hoorn and Kamoun, 2008). One of the interesting results have been reported recently for AVR-Pii and OsExo70-F3 interaction in which OsExo70-F3 physically interacts with AVR-Pii and specifically involved in Pii-dependent resistance suggesting OsExo70-F3 as a helper in Pii/AVR-Pii interactions (Fujisaki et al., 2015). Physical interactions that underlie R/AVR function are starting to be elucidated but still largely ambiguous (Cesari et al., 2013; 2014a; 2014b; Maqbool et al., 2015; Williams et al., 2014). More recently, rice resistant protein pair RGA4/RGA5, was shown to be required for recognition of
Once pathogen effectors are secreted into the host cytoplasm, they induce a reprogramming of host metabolomes (Parker et al., 2009) and transcriptomes (Wei et al., 2013). However, many effectors only partially contribute to virulence except for a few critical effectors known as “core effectors” (Dangl et al., 2013). Some AVR effectors represent typical core effectors and are defined by their substantial contribution to suppression of the plant immune system (Fujisaki et al., 2015; Mackey et al., 2003; Park et al., 2012). In addition to the benefits of AVR effectors, they are also detrimental to the pathogen itself because the detection of AVR effectors by R proteins can halt pathogen proliferation by rapid HR cell death (Greenberg and Yao, 2004). Thus, it will be interesting to reveal the evolutionary and functional advantages of
After a plant senses a pathogen signal, the response leads to activated PTI and subsequent ETI involving a rapid ROS burst (Stael et al., 2015). Scientists have revealed the pivotal role of ROS in the infection response (Levine et al., 1994; Pog?ny et al., 2009; Torres et al., 2005; 2006). As exemplified by the essential role of the
The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase D (NADPH-oxidase D) is a plasma membrane-bound enzyme that generates ROS (Kadota et al., 2015) using the cytosolic electron donor NADPH, supplied by NADP-ME2. NADP-ME2 is a rapidly and abundantly induced enzyme associated with ROS production following
Here, we provide functional evidence that
Rice cultivars Hwayeongbye (HY), Nipponbare (NB), and the
The fungal strain
To analyze the transcriptional level of Os-NADP-ME2-3 in
For the spray inoculation method, incompatible
The coding regions of four alternative splice variants of Os-
To construct the AVR-Pii:mCherry expression cassette, mCherry was cloned into pCB1004 (Yoshida et al., 2009) with
Inverse PCR was performed using two different sets of
The AVR-Pii mutant (AVR-Pii-MT) was obtained by mutation of five negatively charged amino acids (Supplementary Figs. S3A and S3B) using the
The Y2H screening of
Rice leaf sheath preparation, onion tissue preparation, DNA preparation, co-transformation, and biolistic bombardment were performed as described previously (Singh et al., 2012). To detect interactions, 20 μg bait protein (AVR-Pii:pDEST-VYCE?GW) and prey protein (Os-NADP-ME2-3:pDEST-VYNE ?GW) were mixed and bombarded in onion and rice epidermal cells, using biolistic bombardment (Bio-Rad, Biolistic ?-PDS-1000/He Particle Delivery System), as described previously (Singh et al., 2012). They were incubated at 25°C for 12-24 h in the dark followed by cell imaging using a confocal laser microscope (Leica, TCS SP5) at 20× (onion cells) and 40× (rice cells) with a Venus filter (Ex/Em: 488 nm/505-550 nm wavelength). In addition, for the negative control, 20 μg of each AVR-Pii:pDEST-VYCE?GW and pDEST-VYNE?GW; Os-NADP-ME2-3:pDEST-VYNE?GW and pDEST-VYCE?GW; pDEST-VYCE?GW and pDEST-VYNE?GW were bombarded in rice and onion epidermal cells. Similarly,
The 35Sp:4xMyc:AVR-Pii (bait) and 35Sp:3xHA:Os-NADP-ME2-3 (prey) plasmids were generated using the 4xMyc-tagged Co-IP vector pGWB18 and 3xHA-tagged pGWB15, as described previously (Singh et al., 2012). The transient expression assay was performed by infiltrating tobacco leaves with
The N-terminal glutathione S-transferase (GST)-tagged AVR-Pii and AVR-Pii-MT, N-terminal IF2, and 6xHis-tagged Os-NADP-ME2-3 and Os-NADP-ME2-2 were expressed in
Rice varieties HY, NB, and ΔOs-
NADP-ME activity was assayed spectrophotometrically by monitoring NADPH production at 340 nm, using a standard reaction mixture (Detarsio et al., 2004) containing 67 mM Tris-Cl (pH 7.4), 3.5 mM L-malic acid, 0.3 mM NADP+, 5 mM MnCl2, 20 nM Os-NADP-ME2-3 and Os-NADP-ME2-2 in a final volume of 0.7 ml. The reaction was initiated by adding thrombin-treated Os-NADP-ME2-3 and Os-NADP-ME2-2. Initial velocity studies were performed by varying the concentration of one substrate around its Km value, while keeping the other substrate concentration at its saturation level. Kinetic parameters were determined by non-linear regression analysis, and data were fitted to the Michaelis-Menten equation using the curvefitter program of SigmaPlot 10.0 (Erkraft, Germany). Inhibition studies were performed similarly, using 10 nM AVR-Pii and AVR-Pii-MT as inhibitors. The initial velocity studies were performed by varying the concentration of L-malate (0.5-4 mM), while keeping the concentration of NADP+ at its saturation level (0.3 mM) and vice versa. The reaction was started by addition of 20 nM of OsNADP-ME2-3 and 10 nM of AVR-Pii. The competitive inhibition was determined by a double reciprocal plot between 1/Vo and 1/[S]. The dissociation constant of the inhibitors (Ki) was a calculated gradient or intercept term, obtained from the double reciprocal plot. The equation for competitive inhibition is given by
Michaelis constant in the absence of an inhibitor
maximum rate in the absence of an inhibitor
Concentration of an inhibitor
Dissociation constant of the inhibitors
Similarly, the inhibition assays of Os-NADP-ME2-3 by AVR-Pii-MT and Os-NADP-ME2-2 by AVR-Pii were determined by the same protocol. All activity assays were carried out at 25°C using a SP2000UV spectrophotometer (SmartPlus). The protein concentration was determined by the Bio-Rad Protein Assay using BSA as a standard.
ROS production was measured as described previously (Park et al., 2012) with minor modifications. ROS were elicited using chitin and sterile water as a negative control. Briefly, 16 leaf discs from 4 to 5-week-old rice cultivar HY and ΔOs-
Sequence data from this article could be found on the Rice Genome Project website (
We performed Y2H screen using
The interaction between Os-NADP-ME2-3 and AVR-Pii was further validated by bimolecular fluorescence complementation (BiFC) assay, Co-immunoprecipitation (Co-IP) and the glutathione S-transferase (GST) pull-down assay. The BiFC system is based on the formation of a fluorescent complex from the fusion of putative interacting proteins (Gehl et al., 2009). AVR-Pii and Os-NADP-ME2-3 were fused with the C-and N-terminal halves of Venus, then, subjected to the BiFC assay, along with
We found T-DNA insertion mutant rice from Rice Functional Genomic Express Database (RiceGE) developed by the Salk Institute. (
To detect viable invasive hyphae during infection, we produced
We also observed that the entire invaded HY sheath did not experience cell death after infection with
AVR-effectors with native promoters preferentially accumulate in the BIC and translocate into the invaded cytoplasm, then move into neighboring cells, resulting in successful propagation of colonizing hyphae (Khang et al., 2010; Maqbool et al., 2015; Sharma et al., 2013). To analyze the expression pattern of the AVR-Pii effector, we constructed the mCherry-tagged AVR-Pii expression cassette under the control of a native promoter that was recovered by inverse PCR (Supplementary Fig. S8). We inserted the EGFP-tagged putative EHIM matrix protein biotrophy-associated secreted protein (BAS4:EGFP) in front of the AVR-Pii:mCherry cassette (Fig. 4A). BAS4 expression appears as a bright outline in the primary hyphae and inner layer of the BIC (Mosquera et al., 2009). The entire construction was then transformed into
Malic enzyme activity and inhibition studies were performed using purified Os-NADP-ME2-3, Os-NADP-ME2-2, AVR-Pii and AVR-Pii-MT proteins (Supplementary Figs. S10 and S11). From an activity assay, the kinetic properties of both recombinant Os-NADP-ME2-3 and Os-NADP-ME2-2 were calculated. The maximum velocity (Vmax), turnover rate (Kcat) and catalytic efficiency (Kcat/Km) were higher for NADP+ than L-Malate in both cases, whereas these parameters were approximately two-fold higher for the reaction catalyzed by Os-NADP-ME2-3 than by Os-NADP-ME2-2 (Supplementary Table S2). The hyperbolic curve of Os-NADP-ME2-3 activity and its inhibition by AVR-Pii, as well as AVR-Pii-MT, are shown in Figs. 5A and 5B. There was a distinct difference in the activity of Os-NADP-ME2-3 in the absence and presence of the inhibitors (AVR-Pii-MT and AVR-Pii). Shortly after addition of AVR-Pii, the enzyme activity of Os-NADP-ME2-3 was decreased dramatically, while AVR-Pii-MT had no effect (Figs. 5A and 5B). This suggested that AVR-Pii inhibited the activity of Os-NADP-ME2-3, while mutation of AVR-Pii prevented this inhibition. The activity and inhibition data were applied to the Lineweaver-Burk equation (1/activity against 1/[substrate]). This data representation suggested that AVR-Pii competitively inhibited Os-NADP-ME2-3 activity (Fig. 6). Based on the double reciprocal plot, the dissociation constant (Ki) was calculated for both substrates. Ki for NADP+ was higher than that for L-Malate (Supplementary Table S3). Next, Os-NADP-ME2-2 showed extremely lower activity than that of Os-NADP-ME2-3. In addition, there was no inhibition of Os-NADP-ME2-2 by AVR-Pii (Supplementary Figs. S12 and S13). These results demonstrate that AVR-Pii but not AVR-Pii-MT specifically inhibits
Chitin is known fungal PAMP elicitor that generates ROS after its perception by plant cells. Rice cells can perceive chitin through pattern recognition receptor CEBiP/OsCERK1 (Shimizu et al., 2010). To determine whether ROS generation is affected in
Our data provide evidence that AVR-Pii-triggered ROS inhibition (ATRI) helps
Insights into the function of secreted fungal effectors in the reprogramming of host defense and metabolism are gradually emerging. Given the plethora of secreted proteins encoded in the genomes of phytopathogenic fungi, only a few fungal effector targets have been identified to date, with less than a handful shown to impair host metabolic functions directly (Djamei et al., 2011; Hemetsberger et al., 2012; Tanaka et al., 2014). Although eight
The Y2H, BiFC, Co-IP and GST pull-down data of AVR-Pii and Os-NADP-ME2-3 strongly support the notion that AVR-Pii interacts with Os-NADP-ME2-3 in the rice cytoplasm (Figs. 1 and 2; Supplementary Fig. S2). This is in agreement with the data from Parker et al. (2009), whereby Os-NADP-MEs activities are localized at the penetration site following
A decrease in ROS accumulation occurred in susceptible cultivar at 24 h post-inoculation (hpi), but no change in ROS concentration was observed in resistant cultivar following
AVR effectors have dual activities: they suppress PTI thereby enhancing pathogenesis in host cells in the absence of its cognate R genes, and they trigger rapid HR in the presence of R proteins by ETI (G?hre and Robatzek, 2008). OsExo70-F2 which is also identified in our study as AVR-Pii interacting protein has been suggested as an additional factor in ETI immunity by interacting with AVR-Pii (Fujisaki et al., 2015).We observed distinct AVR-Pii expression as well as typical rice blast symptoms (Figs. 3B and 4B), as expected from its virulence function in susceptible rice cultivar NB (Fujisaki et al., 2015). However, in the ΔOs-
Our data agree with previous OsRac1-mediated ROS production and cell death mechanisms (Kawasaki et al., 1999), which involve a positive regulator of resistance to fungal and bacterial pathogens (Ono et al., 2001). Rice OsRac1 regulates ROS production by directly interacting with the N-terminus of NADPH-oxidase (Wong et al., 2004) and forming a putative immune complex at the plasma membrane with RACK1A, RAR1, SGT1, HSP90, and HSP70 (Nakashima et al., 2008). The interaction of tobacco NADP-ME with HSP70 (Lara et al., 2005) suggests that Os-NADP-ME2 interacts with Nakashima’s immune complexes (Nakashima et al., 2008), which were recently described as a defensome (Akamatsu et al., 2013). This indicates that Os-NADP-ME2 can directly regulate ROS production in the defensome complex to control innate immunity. Recent data support that massive ROS bursts could inactivate infecting
Based on our results, we generated a model of AVR-Pii and Os-NADP-ME interactions near the BIC after infection. In our model,
Mol. Cells 2016; 39(5): 426-438
Published online May 31, 2016 https://doi.org/10.14348/molcells.2016.0094
Copyright © The Korean Society for Molecular and Cellular Biology.
Raksha Singh1,2,3, Sarmina Dangol1,3, Yafei Chen1,3, Jihyun Choi1, Yoon-Seong Cho1, Jea-Eun Lee1, Mi-Ok Choi1, and Nam-Soo Jwa1,*
1Division of Integrative Bioscience and Biotechnology, College of Life Sciences, Sejong University, Seoul 143-747, Korea, 2Present address: Horticulture section, School of Integrative Plant Science, Cornell University, New York State Agricultural Experiment Station, Geneva, New York, United States of America,
Correspondence to:*Correspondence: nsjwa@sejong.ac.kr
Plant disease resistance occurs as a hypersensitive response (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant immune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the
Keywords: AVR-effectors, gene-for-gene interaction, NADP-Malic enzyme, reactive oxygen species, rice
Many scientists have striven to find ways to control hemibiotrophic
Pathogen AVR effectors have been genetically proven to be essential components in plant immune responses (Flor, 1971). However, the mechanism by which AVR effectors and R proteins are associated with these responses remains unclear. Initially, the ligand-receptor model (Gabriel and Rolfe, 1990) was widely supported, but the lack of physical interactions between a number of R/AVR pairs has resulted in the generation of alternative guard and decoy hypothesis (McHale et al., 2006; van der Hoorn and Kamoun, 2008). One of the interesting results have been reported recently for AVR-Pii and OsExo70-F3 interaction in which OsExo70-F3 physically interacts with AVR-Pii and specifically involved in Pii-dependent resistance suggesting OsExo70-F3 as a helper in Pii/AVR-Pii interactions (Fujisaki et al., 2015). Physical interactions that underlie R/AVR function are starting to be elucidated but still largely ambiguous (Cesari et al., 2013; 2014a; 2014b; Maqbool et al., 2015; Williams et al., 2014). More recently, rice resistant protein pair RGA4/RGA5, was shown to be required for recognition of
Once pathogen effectors are secreted into the host cytoplasm, they induce a reprogramming of host metabolomes (Parker et al., 2009) and transcriptomes (Wei et al., 2013). However, many effectors only partially contribute to virulence except for a few critical effectors known as “core effectors” (Dangl et al., 2013). Some AVR effectors represent typical core effectors and are defined by their substantial contribution to suppression of the plant immune system (Fujisaki et al., 2015; Mackey et al., 2003; Park et al., 2012). In addition to the benefits of AVR effectors, they are also detrimental to the pathogen itself because the detection of AVR effectors by R proteins can halt pathogen proliferation by rapid HR cell death (Greenberg and Yao, 2004). Thus, it will be interesting to reveal the evolutionary and functional advantages of
After a plant senses a pathogen signal, the response leads to activated PTI and subsequent ETI involving a rapid ROS burst (Stael et al., 2015). Scientists have revealed the pivotal role of ROS in the infection response (Levine et al., 1994; Pog?ny et al., 2009; Torres et al., 2005; 2006). As exemplified by the essential role of the
The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase D (NADPH-oxidase D) is a plasma membrane-bound enzyme that generates ROS (Kadota et al., 2015) using the cytosolic electron donor NADPH, supplied by NADP-ME2. NADP-ME2 is a rapidly and abundantly induced enzyme associated with ROS production following
Here, we provide functional evidence that
Rice cultivars Hwayeongbye (HY), Nipponbare (NB), and the
The fungal strain
To analyze the transcriptional level of Os-NADP-ME2-3 in
For the spray inoculation method, incompatible
The coding regions of four alternative splice variants of Os-
To construct the AVR-Pii:mCherry expression cassette, mCherry was cloned into pCB1004 (Yoshida et al., 2009) with
Inverse PCR was performed using two different sets of
The AVR-Pii mutant (AVR-Pii-MT) was obtained by mutation of five negatively charged amino acids (Supplementary Figs. S3A and S3B) using the
The Y2H screening of
Rice leaf sheath preparation, onion tissue preparation, DNA preparation, co-transformation, and biolistic bombardment were performed as described previously (Singh et al., 2012). To detect interactions, 20 μg bait protein (AVR-Pii:pDEST-VYCE?GW) and prey protein (Os-NADP-ME2-3:pDEST-VYNE ?GW) were mixed and bombarded in onion and rice epidermal cells, using biolistic bombardment (Bio-Rad, Biolistic ?-PDS-1000/He Particle Delivery System), as described previously (Singh et al., 2012). They were incubated at 25°C for 12-24 h in the dark followed by cell imaging using a confocal laser microscope (Leica, TCS SP5) at 20× (onion cells) and 40× (rice cells) with a Venus filter (Ex/Em: 488 nm/505-550 nm wavelength). In addition, for the negative control, 20 μg of each AVR-Pii:pDEST-VYCE?GW and pDEST-VYNE?GW; Os-NADP-ME2-3:pDEST-VYNE?GW and pDEST-VYCE?GW; pDEST-VYCE?GW and pDEST-VYNE?GW were bombarded in rice and onion epidermal cells. Similarly,
The 35Sp:4xMyc:AVR-Pii (bait) and 35Sp:3xHA:Os-NADP-ME2-3 (prey) plasmids were generated using the 4xMyc-tagged Co-IP vector pGWB18 and 3xHA-tagged pGWB15, as described previously (Singh et al., 2012). The transient expression assay was performed by infiltrating tobacco leaves with
The N-terminal glutathione S-transferase (GST)-tagged AVR-Pii and AVR-Pii-MT, N-terminal IF2, and 6xHis-tagged Os-NADP-ME2-3 and Os-NADP-ME2-2 were expressed in
Rice varieties HY, NB, and ΔOs-
NADP-ME activity was assayed spectrophotometrically by monitoring NADPH production at 340 nm, using a standard reaction mixture (Detarsio et al., 2004) containing 67 mM Tris-Cl (pH 7.4), 3.5 mM L-malic acid, 0.3 mM NADP+, 5 mM MnCl2, 20 nM Os-NADP-ME2-3 and Os-NADP-ME2-2 in a final volume of 0.7 ml. The reaction was initiated by adding thrombin-treated Os-NADP-ME2-3 and Os-NADP-ME2-2. Initial velocity studies were performed by varying the concentration of one substrate around its Km value, while keeping the other substrate concentration at its saturation level. Kinetic parameters were determined by non-linear regression analysis, and data were fitted to the Michaelis-Menten equation using the curvefitter program of SigmaPlot 10.0 (Erkraft, Germany). Inhibition studies were performed similarly, using 10 nM AVR-Pii and AVR-Pii-MT as inhibitors. The initial velocity studies were performed by varying the concentration of L-malate (0.5-4 mM), while keeping the concentration of NADP+ at its saturation level (0.3 mM) and vice versa. The reaction was started by addition of 20 nM of OsNADP-ME2-3 and 10 nM of AVR-Pii. The competitive inhibition was determined by a double reciprocal plot between 1/Vo and 1/[S]. The dissociation constant of the inhibitors (Ki) was a calculated gradient or intercept term, obtained from the double reciprocal plot. The equation for competitive inhibition is given by
Michaelis constant in the absence of an inhibitor
maximum rate in the absence of an inhibitor
Concentration of an inhibitor
Dissociation constant of the inhibitors
Similarly, the inhibition assays of Os-NADP-ME2-3 by AVR-Pii-MT and Os-NADP-ME2-2 by AVR-Pii were determined by the same protocol. All activity assays were carried out at 25°C using a SP2000UV spectrophotometer (SmartPlus). The protein concentration was determined by the Bio-Rad Protein Assay using BSA as a standard.
ROS production was measured as described previously (Park et al., 2012) with minor modifications. ROS were elicited using chitin and sterile water as a negative control. Briefly, 16 leaf discs from 4 to 5-week-old rice cultivar HY and ΔOs-
Sequence data from this article could be found on the Rice Genome Project website (
We performed Y2H screen using
The interaction between Os-NADP-ME2-3 and AVR-Pii was further validated by bimolecular fluorescence complementation (BiFC) assay, Co-immunoprecipitation (Co-IP) and the glutathione S-transferase (GST) pull-down assay. The BiFC system is based on the formation of a fluorescent complex from the fusion of putative interacting proteins (Gehl et al., 2009). AVR-Pii and Os-NADP-ME2-3 were fused with the C-and N-terminal halves of Venus, then, subjected to the BiFC assay, along with
We found T-DNA insertion mutant rice from Rice Functional Genomic Express Database (RiceGE) developed by the Salk Institute. (
To detect viable invasive hyphae during infection, we produced
We also observed that the entire invaded HY sheath did not experience cell death after infection with
AVR-effectors with native promoters preferentially accumulate in the BIC and translocate into the invaded cytoplasm, then move into neighboring cells, resulting in successful propagation of colonizing hyphae (Khang et al., 2010; Maqbool et al., 2015; Sharma et al., 2013). To analyze the expression pattern of the AVR-Pii effector, we constructed the mCherry-tagged AVR-Pii expression cassette under the control of a native promoter that was recovered by inverse PCR (Supplementary Fig. S8). We inserted the EGFP-tagged putative EHIM matrix protein biotrophy-associated secreted protein (BAS4:EGFP) in front of the AVR-Pii:mCherry cassette (Fig. 4A). BAS4 expression appears as a bright outline in the primary hyphae and inner layer of the BIC (Mosquera et al., 2009). The entire construction was then transformed into
Malic enzyme activity and inhibition studies were performed using purified Os-NADP-ME2-3, Os-NADP-ME2-2, AVR-Pii and AVR-Pii-MT proteins (Supplementary Figs. S10 and S11). From an activity assay, the kinetic properties of both recombinant Os-NADP-ME2-3 and Os-NADP-ME2-2 were calculated. The maximum velocity (Vmax), turnover rate (Kcat) and catalytic efficiency (Kcat/Km) were higher for NADP+ than L-Malate in both cases, whereas these parameters were approximately two-fold higher for the reaction catalyzed by Os-NADP-ME2-3 than by Os-NADP-ME2-2 (Supplementary Table S2). The hyperbolic curve of Os-NADP-ME2-3 activity and its inhibition by AVR-Pii, as well as AVR-Pii-MT, are shown in Figs. 5A and 5B. There was a distinct difference in the activity of Os-NADP-ME2-3 in the absence and presence of the inhibitors (AVR-Pii-MT and AVR-Pii). Shortly after addition of AVR-Pii, the enzyme activity of Os-NADP-ME2-3 was decreased dramatically, while AVR-Pii-MT had no effect (Figs. 5A and 5B). This suggested that AVR-Pii inhibited the activity of Os-NADP-ME2-3, while mutation of AVR-Pii prevented this inhibition. The activity and inhibition data were applied to the Lineweaver-Burk equation (1/activity against 1/[substrate]). This data representation suggested that AVR-Pii competitively inhibited Os-NADP-ME2-3 activity (Fig. 6). Based on the double reciprocal plot, the dissociation constant (Ki) was calculated for both substrates. Ki for NADP+ was higher than that for L-Malate (Supplementary Table S3). Next, Os-NADP-ME2-2 showed extremely lower activity than that of Os-NADP-ME2-3. In addition, there was no inhibition of Os-NADP-ME2-2 by AVR-Pii (Supplementary Figs. S12 and S13). These results demonstrate that AVR-Pii but not AVR-Pii-MT specifically inhibits
Chitin is known fungal PAMP elicitor that generates ROS after its perception by plant cells. Rice cells can perceive chitin through pattern recognition receptor CEBiP/OsCERK1 (Shimizu et al., 2010). To determine whether ROS generation is affected in
Our data provide evidence that AVR-Pii-triggered ROS inhibition (ATRI) helps
Insights into the function of secreted fungal effectors in the reprogramming of host defense and metabolism are gradually emerging. Given the plethora of secreted proteins encoded in the genomes of phytopathogenic fungi, only a few fungal effector targets have been identified to date, with less than a handful shown to impair host metabolic functions directly (Djamei et al., 2011; Hemetsberger et al., 2012; Tanaka et al., 2014). Although eight
The Y2H, BiFC, Co-IP and GST pull-down data of AVR-Pii and Os-NADP-ME2-3 strongly support the notion that AVR-Pii interacts with Os-NADP-ME2-3 in the rice cytoplasm (Figs. 1 and 2; Supplementary Fig. S2). This is in agreement with the data from Parker et al. (2009), whereby Os-NADP-MEs activities are localized at the penetration site following
A decrease in ROS accumulation occurred in susceptible cultivar at 24 h post-inoculation (hpi), but no change in ROS concentration was observed in resistant cultivar following
AVR effectors have dual activities: they suppress PTI thereby enhancing pathogenesis in host cells in the absence of its cognate R genes, and they trigger rapid HR in the presence of R proteins by ETI (G?hre and Robatzek, 2008). OsExo70-F2 which is also identified in our study as AVR-Pii interacting protein has been suggested as an additional factor in ETI immunity by interacting with AVR-Pii (Fujisaki et al., 2015).We observed distinct AVR-Pii expression as well as typical rice blast symptoms (Figs. 3B and 4B), as expected from its virulence function in susceptible rice cultivar NB (Fujisaki et al., 2015). However, in the ΔOs-
Our data agree with previous OsRac1-mediated ROS production and cell death mechanisms (Kawasaki et al., 1999), which involve a positive regulator of resistance to fungal and bacterial pathogens (Ono et al., 2001). Rice OsRac1 regulates ROS production by directly interacting with the N-terminus of NADPH-oxidase (Wong et al., 2004) and forming a putative immune complex at the plasma membrane with RACK1A, RAR1, SGT1, HSP90, and HSP70 (Nakashima et al., 2008). The interaction of tobacco NADP-ME with HSP70 (Lara et al., 2005) suggests that Os-NADP-ME2 interacts with Nakashima’s immune complexes (Nakashima et al., 2008), which were recently described as a defensome (Akamatsu et al., 2013). This indicates that Os-NADP-ME2 can directly regulate ROS production in the defensome complex to control innate immunity. Recent data support that massive ROS bursts could inactivate infecting
Based on our results, we generated a model of AVR-Pii and Os-NADP-ME interactions near the BIC after infection. In our model,
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