The Nup98 Homolog APIP12 Targeted by the Effector AvrPiz-t is Involved in Rice Basal Resistance Against Magnaporthe oryzae
© The Author(s). 2017
Received: 21 October 2016
Accepted: 10 February 2017
Published: 15 February 2017
The effector AvrPiz-t of Magnaporthe oryzae has virulence function in rice. However, the mechanism underlying its virulence in host is not fully understood.
In this study, we analyzed the function of AvrPiz-t interacting protein 12 (APIP12) in rice immunity. APIP12 significantly bound to AvrPiz-t and APIP6 in its middle portion and N-terminus, respectively, in yeast two-hybrid assay. Glutathione S-transferase (GST) pull-down assay further verified the interactions of APIP12 with AvrPiz-t and APIP6. APIP12 encodes a homologue of nucleoporin protein Nup98 without the conserved domain of Phe-Gly repeats and has no orthologue in other plants. Both knockout and knockdown of APIP12 caused enhanced susceptibility of rice plants to virulent isolates of M. oryzae. The expression of some pathogenesis-related (PR) genes was reduced in both knockout and knockdown mutants, suggesting that APIP12 is required for the accumulation of transcripts of PR genes upon the infection. It is worth noting that neither knockout/knockdown nor overexpression of APIP12 attenuates Piz-t resistance.
Taken together, our results demonstrate that APIP12 is a virulence target of AvrPiz-t and is involved in the basal resistance against M. oryzae in rice.
KeywordsAPIP12 AvrPiz-t Protein-protein interaction Blast resistance Nup98
Plants possess a two-layer innate immune system to protect themselves from most microbial pathogens (Dodds and Rathjen 2010; Jones and Dangl 2006). The first layer of innate immunity is called PAMP-triggered immunity (PTI) that the recognition of pathogen-associated molecular patterns (PAMPs) is mounted by the pattern recognition receptors (PRRs) (Jones and Dangl 2006). PTI comprises the accumulation of reactive oxygen species (ROS) and the deposition of phenolic compounds (Jones and Dangl 2006). However, some pathogens are able to suppress the first layer of resistance by the secretion of effector proteins to target PRRs or key components in the PTI signaling pathway. Plants have evolved the second layer of the immune system called effector-triggered immunity (ETI), which is activated upon recognition of pathogen-secreted avirulence (Avr) effectors by cognate plant resistance (R) receptors (Bonardi and Dangl 2012). The resistance mediated by ETI is often associated with a hypersensitive response (HR) at the infection site to inhibit pathogen proliferation (Chisholm et al. 2006; Jones and Dangl 2006). Effectors are highly variable among different strains of a pathogen species. Thus, compared with PTI, ETI is more specific and assumed to be less durable.
The interaction between rice and Magnaporthe oryzae, the fungal pathogen causing the devastating rice blast disease, has been considered as one of the model phytopathosystems for understanding PTI and ETI in plant-fungal interactions (Dean et al. 2012; Liu et al. 2013). Over 25 rice R genes to blast have been molecularly characterized and most of them encode R proteins containing nucleotide binding site (NBS) and leucine-rich repeats (LRR) domains (Leung et al. 2015). In parallel with the advances in characterization of host R genes, over 10 Avr genes cognate to rice R genes have been characterized in M. oryzae (Wu et al. 2015; Zhang et al. 2015b). Two distinct models have been illustrated for the recognition of rice R proteins to their cognate M. oryzae Avr proteins. In the case of Pita/AvrPita, Pik/AvrPik, Pi-CO39/Avr1-CO39, and Pia/AvrPia, R proteins physically bind to their cognate Avr proteins (Cesari et al. 2013; Dangl and Jones 2001; Kanzaki et al. 2012). On the contrary, no direct interactions has been either identified or documented in other R/Avr pairs, e.g., no binding activity between Pi9 and AvrPi9 observed in a yeast two-hybrid (Y2H) assay (Wu et al. 2015). In the case of indirect interactions, recognition between R and Avr proteins is assumed to be activated by either so-called “guardees” or “decoys” (van der Hoorn and Kamoun 2008). In this regard, functional characterization of the host targets operated by Avr genes is vital for the elucidation of the recognition between R and Avr genes and the subsequent activation of resistance signaling.
We previously characterized the AvrPiz-t gene cognate to the rice blast R gene Piz-t using a map-based cloning strategy in M. oryzae (Li et al. 2009). Ectopic expression of AvrPiz-t in both tobacco and rice can suppress BAX-mediated cell death and PTI activated by both chitin and flg22, respectively, indicating that AvrPiz-t could primarily function as a virulence effector promoting pathogenicity without the Piz-t gene in the host (Li et al. 2009; Park et al. 2012). By Y2H screening, we identified 12 AvrPiz-t interacting proteins (APIPs) in rice. Functional characterization of APIP6 revealed that AvrPiz-t can suppress the E3 ligase activity of APIP6, which is required for the basal resistance to blast in rice (Park et al. 2012). In this study, we aim to characterize the function of APIP12 which encodes a protein homologous to nucleoporin 98 (Nup98). As essential components required for the assembly of nuclear pore complex (NPC), Nups play important roles in plant immunity. In Arabidopsis, both Nup160 and Nup96 were found to be involved in immunity-related mRNA export (Wiermer et al. 2012; Zhang and Li 2005). In this study, we will employ both Y2H and GST pull-down assays to validate and determine the regions required for direct binding between AvrPiz-t and APIP12. The interaction between APIP12 and other APIPs will also be tested to investigate whether different APIPs are functioning in a complex. The function of APIP12 in the basal and Piz-t-mediated resistance to rice blast will then be genetically characterized using knockout/knockdown mutants of APIP12.
APIP12 encodes a unique protein sharing sequence and structure similarity to the Nup98 protein family
APIP12 employs different regions to interact with AvrPiz-t and APIP6
Next, we employed glutathione S-transferase (GST) pull-down assay to verify the interaction between APIP12 and AvrPiz-t in vitro. Immunoblotting analysis using the anti-GST and anti-MBP antibodies indicated a proper load of GST, GST-fused different portions of APIP12, and MBP-AvrPiz-t in the input (Fig. 2b). After pull-down procedure, the GST and 3 GST-fused APIP12s in the elution were able to be detected by the anti-GST antibody (Fig. 2b). Moreover, MBP-AvrPiz-t was detected in the elution from each of mixtures of GST-fused APIP12s and MBP-AvrPiz-t by anti-MBP antibody (Fig. 2b). On the contrary, no signal was detected in the one of GST incubates without APIP12 (Fig. 2b). These data clearly indicated that AvrPiz-t and APIP12 interacted to each other in GST pull-down assay. It is worth noting that the signal intensity of GST-APIP12s was much lower than the one of GST alone although equal amount of proteins were loaded in the input (Fig. 2b). Moreover, additional protein products with smaller sizes of GST-fused APIP12F and APIP12M displayed GST signal in both input and elution without a known reason (Fig. 2b).
To investigate whether APIP12 could form a complex with APIP6, we tested their interaction using both Y2H and GST pull-down assays. As Fig. 2c illustrated, positive signals were observed when either APIP12F or N was co-transformed with APIP6 (Fig. 2c). In GST pull-down assay, APIP6 was detected in the GST incubates of APIP12F (Fig. 2d). However, we could not detect the deposition of APIP6 in the GST incubates of APIP12N, displaying a distinct interaction manner from the one displayed in Y2H (Fig. 2c and d). In addition, we did not identify the interaction between APIP12M and APIP6 in either Y2H or pull-down assays, suggesting that APIP12M is not responsible for the binding of APIP12 to APIP6 (Fig. 2c and d). On the contrary, APIP12-M was found to be the major region binding to AvrPiz-t (Fig. 2a and b). These data prompted us to propose that APIP12 and APIP6 work as a protein complex involved in the basal resistance to rice blast.
The interaction between APIP12 and Piz-t was also investigated to check whether they can form complex by physical binding. Contrasting to the significant growth on the –Leu/-Trp medium, no growth was observed of the yeast cells co-expressed with different portions of Piz-t and APIP12 on the selective medium (Additional file 1: Figure S1). This data suggested that APIP12 might not physically bind to Piz-t.
APIP12 is involved in the basal resistance against rice blast
To functionally characterize APIP12 in the basal resistance, a Tos17 insertion line, RMD_TosRS-04Z11BB04 (APIP12-KO) in rice variety ZH11 was identified and validated. The Tos17 is situated at 476-bp upstream from the start codon of APIP12, which was confirmed by the DNA analysis using specific primers (Additional file 2: Figure S2). RT-PCR analysis revealed that the expression of APIP12 was undetectable in the insertion line. On the contrary, the wild type plant showed a weak but significant level of expression of APIP12 (Additional file 2: Figure S2). We thus speculate that insertion of Tos17 successfully interrupts the gene expression of APIP12, providing an ideal genetic material for its functional characterization. Both ZH11 and APIP12-KO were inoculated with the virulent isolate GUY11. The mean lesion density in ZH11 was 5.07/3 cm2 whereas the one in APIP12-KO was 11.50/3 cm2 (Fig. 3b and c), indicating that disease symptom in the mutant plant is much severer than the one in wild type plant. In addition to the spray method, we also quantified the resistance of rice lines using the punch method. The mean lesion size in APIP12-KO was 12.98 mm2, which was significantly larger than the one of 9.37 mm2 in ZH11 (Fig. 3d and e). We further quantified disease resistance of APIP12 knockdown (APIP12-KD) rice lines generated via RNAi. Compared to the one in the wild type plant, the expression of APIP12 is significantly reduced in both APIP12-KD lines (46-2 and 49-2, Additional file 3: Figure S3a). The lesion size observed in these two KD lines was much larger than the one in the wild type plant, indicating that they are more susceptible to the virulent isolate (Additional file 3: Figure S3b and c). Taken together, we conclude that either knockout or knockdown of APIP12 resulted in more susceptibility of rice plants to rice blast.
In addition to the knockout/knockdown mutants, 3 independent lines of APIP12 overexpression (APIP12-OX-69, −70, and −73) validated by gene expression (Additional file 4: Figure S4a) were challenged with the virulent isolate KJ201 by both spray and punch inoculations. No significant difference in disease resistance was observed in APIP12-OX lines compared to the non-transgenic NPB plant (Additional file 4: Figure S4b), suggesting that overexpression of APIP12 did not alter the resistance to rice blast.
APIP12 is not required for the Piz-t-mediate resistance to rice blast
The expression of PR genes is subdued in APIP12-knockout/knockdown mutants upon the infection of rice blast
Relatively expression level of PR genes in the APIP12-knockout mutant (M) compared to the one in the wild type plant (W) at 72 h after infection (HPI) with the virulent isolate GUY11
PR gene family
Relative expression level (M/W)
0.45 ± 0.090***
0.53 ± 0.177**
0.93 ± 0.261
1.37 ± 0.440
0.60 ± 0.089**
0.68 ± 0.018***
0.97 ± 0.105
0.57 ± 0.055***
0.71 ± 0.089
0.63 ± 0.132**
0.22 ± 0.116***
1.39 ± 0.218
0.51 ± 0.060***
0.33 ± 0.043***
0.71 ± 0.324
0.65 ± 0.150
0.59 ± 0.189
0.47 ± 0.102***
1.03 ± 0.604
0.34 ± 0.080***
0.17 ± 0.033***
0.24 ± 0.038***
0.37 ± 0.051***
Multiple lines of evidence have demonstrated that phytopathogens secrete and deliver diverse effectors into plant cells to interfere with individual defense responses (Gohre and Robatzek 2008). It has been also clearly illustrated that in the absence of its cognate resistance protein, the so-called avirulence effector fulfills the virulence function to promote the pathogenicity of the pathogen (Dou and Zhou 2012). As described previously, AvrPiz-t can suppress the BAX-mediated cell death in tobacco leaves and the generation of reactive oxygen species (ROS) induced by PAMP elicitors in rice (Li et al. 2009; Park et al. 2012), demonstrating the virulence activity of AvrPiz-t in the absence of its cognate resistance gene Piz-t. Likewise, the RXLR effector AVR1 of Phytophthora infestants functions as a virulence factor that promotes colonization and suppresses callose deposition, a hallmark of basal defense in tobacco (Du et al. 2015). It has been extensively illustrated that many phytopathogen effector proteins execute the virulence activity through the manipulation of their host targets. For example, Pseudomonas syringae type III effector HopF2Pto targets Arabidopsis RIN4 protein to promote the growth of bacterium in plant (Wilton et al. 2010). The effector Avr3a essential for the virulence of P. infestants targets and stabilizes the plant E3 ligase CMPG1 for potentially preventing host cell death during the biotrophic phase of infection (Bos et al. 2010). The identification of 12 APIPs targeted by AvrPiz-t allows the dissection of how AvrPiz-t contributes its virulence functionality to rice blast pathogen. Park et al. (2012) demonstrated that AvrPiz-t could target APIP6 and suppress its E3 ligase activity during the infection process. Moreover, APIP6 is required for PTI and its silencing leads to the compromised resistance to rice blast. These data provided the evidence that AvrPiz-t conveys its virulence advantage by manipulating APIP6 that is involved in the basal resistance against rice blast. Most recently, the study on APIP10, which encodes another E3 ligase, elucidated a similar mechanism on how AvrPiz-t interacts with host target and promotes its virulence to rice blast (Park et al. 2016). Most recently, AvrPiz-t was demonstrated to suppress the transcriptional activity and protein accumulation of APIP5 for promoting the effector-triggered necrosis in rice (Wang et al., 2016).
In this study, we introduce APIP12, the 4th host protein targeted by AvrPiz-t and attempt to illustrate its role in rice immunity against rice blast. APIP12 displays an induced expression pattern in both compatible and incompatible reactions, suggesting that it responds positively to rice blast infection. Either knockout or knockdown of APIP12 in rice led to the enhanced susceptibility to rice blast. On the contrary, the mutation of APIP12 did not compromise the immunity to rice blast mediated by the pair of Piz-t and AvrPiz-t. These data indicated that APIP12 is most likely not required for the Piz-t-mediated resistance. Nevertheless, it functions as a positive regulator in the basal resistance against rice blast. The attenuated induced expression of PR genes in the compatible interaction to rice blast in APIP12 mutants further supported this hypothesis. It is thus reasonable to speculate that APIP12 is one of the virulence targets, like APIP6 and APIP10, and manipulated by AvrPiz-t for executing its virulence function in rice (Park et al. 2012; Park et al. 2016). Moreover, the interaction assay in yeast clearly indicated that APIP12 employed different domains to interact with AvrPiz-t and APIP6, providing a clue of the connection among these 3 proteins. However, it is still elusive how AvrPiz-t contributes its virulence to rice blast in rice through the modification of APIP12.
Increasing evidence points the notion that some components of NPC are involved in the regulation of immunity to diverse pathogens by selectively regulating the exchange of proteins and RNAs of components in plant defense signaling. For example, in the largest subunit of the NPC, 3 out of 9 putative complex members of the Nup107-160 sub-complex, i.e., MOS3/Nup96, Nup160, and Seh1, were found to be essential for the basal resistance and snc1 activated autoimmunity in Arabidopsis (Du et al. 2009; Ratner et al. 2007). Given the importance of NUPs in disease resistance, we assume that they could also be targeted by pathogen effectors like those components in defense signaling from an evolutionary point of view (Gohre and Robatzek 2008). The existence of two Nup98 featured domains (GLEBS and nucleoporin2) in APIP12 promoted us to speculate that it is a component of NPC in rice. The interaction between APIP12 with OsNup96 (LOC_03g07580) was identified by Y2H assay (Additional file 6: Figure S5), providing additional evidence to the association between APIP12 with other components in NPC as characterized previously (Ratner et al. 2007). However, we found that APIP12 is present in the cytoplasmic foci rather than in the nuclei or the nuclear envelope to which typical NUPs are localized (Boeglin et al. 2016; Zhang and Li 2005). Moreover, distinct from other versions of Nup98s identified in all branches of eukaryotic life including rice containing the FG domain (Schmidt and Gorlich 2015), APIP12 does not contain this conserved domain. The FG domain was documented to be important for the binding to the constituents of the NPC scaffold. For example, the binding of the FG domain of Nup98 with Nup93 is important for the assembly of FG domain in the center of the NPC (Xu and Powers 2013). More importantly, the FG domain could mainly function in the formation of the selective permeability barrier of NPC by its spontaneous phase-separation from aqueous solution into FG particles (Robles et al. 2012; Xu and Powers 2013). Intriguingly, APIP12 is present only in rice but not in other monocot and dicot plants. Taken together, we postulate that APIP12 could represent a unique Nup98 homologue involved in the basal resistance to rice blast.
Plant materials and growth conditions
Rice (Oryza sativa L.) lines, including Nipponbare (NPB), Zhonghua11 (ZH11), the Piz-t transgenic line in NPB (NPB-Piz-t) as described previously (Li et al. 2009), are used in this study. A Tos17 insertion line of the APIP12 gene in ZH11, TosRs_04Z11BB04, is obtained from the Rice Mutant Database (http://rmd.ncpgr.cn/). Rice plants are grown in pots and kept in a growth chamber ranging from 22 to 26 °C with the cycle of 14 h light/10 h dark.
Plasmid construction and rice transformation
To generate the RNAi vector of APIP12, a 339-bp fragment of at the C-terminus (1,261 and 1,600 bp downstream of the start codon) was amplified and cloned into the pANDA vector (Miki and Shimamoto 2004) by LR gateway reactions. The APIP12 overexpression vector was constructed in pCambia1301 in which the full-length coding sequence (CDS) of APIP12 was inserted downstream of the maize (Zea mays) ubiquitin promoter. All the primers are given in Additional file 5: Table S1. The vectors were transformed into NPB to generate transgenic lines via the Agrobacterium-mediated rice transformation method described previously (Qu et al. 2006). Over 20 independent transgenic lines were generated and used for functional characterization.
Rice blast inoculation and disease resistance evaluation
The M. oryzae isolates GUY11, KJ201, and GUY11-AvrPiz-t transformed strain (Li et al. 2009) were used for the rice blast inoculation. The fungus was cultured on complete medium (CM) for 2 weeks at 28 °C. Three-week old rice seedlings were inoculated with the conidial suspensions at concentration of 10-15×104 spores ml−1 with a sprayer (Qu et al. 2006). Disease assessment was conducted 7 days after inoculation. The experiments were repeated for two times. In addition to the spray method, the punch method was employed to quantify the blast resistance of 6-week old plants as described previously (Ono et al. 2001). Mean size of lesions was measured using Image J software (https://imagej.nih.gov/ij/). The Student’s T-Test was used to determine the significance of differences between mutant and wild type.
To identify Nup98 homologues in different plant species, homolog search against the NCBI non-redundant (NR) protein database (http://blast.ncbi.nlm.nih.gov/) was performed using APIP12 as a query. Accession numbers of the homologues are as follows: LOC_Os12g06870 and LOC_ Os12g06890 (Oryza sativa, identified from rice genome database: http://rice.plantbiology.msu.edu/), NP_172510 (Arabidopsis thaliana), BAF98996 (Daucus carota), XP_010660457 (Vitis vinifera), XP_002317654 (Populus trichocarpa), XP_008673404 (Zea mays), BAK00061 (Hordeum vulgare), and XP_002446695 (Sorghum bicolor). Protein sequences were aligned using the Clustalw2 program (http://www.ebi.ac.uk/Tools/msa/clustalw2/) with the default parameters. The phylogenetic tree was viewed by using the neighbor-joining method of the MEGA 4.1 software (Tamura et al. 2007). A bootstrap analysis with 1000 replicates was performed to test the confidence of topology. We included Nup98 in human as an outgroup (Accession No. : AAH41136) in the phylogenetic analysis.
Yeast two-hybrid analysis
The Y2H assay was performed by following the procedure as described previously (Park et al. 2012) using the ProQuest Two-Hybrid system (Invitrogen, USA). The mature form of AvrPiz-t (residues 19–108) was cloned into pDBLeu-BD to generate the BD:Ns-AvrPiz-t bait construct. The full length, N-terminus, middle portion (M) and C-terminus of APIP12 were cloned into pPC86-AD to generate the AD:APIP12F, AD:APIP12N, AD:APIP12M and AD:APIP12C prey constructs, respectively. All the primers are given in Additional file 5: Table S1. Yeast cells with co-transformed pDBLeu- and pPC86-derived vectors were plated and incubated on synthetic medium lacking leucine, tryptophan (DOB-Leu-Trp) and the selective medium DOB-Leu-Trp-His (supplemented with 50 mM of 3-amino-1,2,4-triazole) at 30 °C for 3 days for the observation of cell growth to detect the His reporter gene activity. The yeast colonies gowning on DOB-Leu-Trp medium were blotted onto filter papers. The filter papers were frozen in liquid nitrogen for 1 min, and then were placed on top of a pre-soaked filter paper in a 100 mm sterile plate with Z buffer/X-gal solution [2 ml of Z buffer, 6 μl ofβ-mercaptoethanol, 20 μl of X-gal stock solution (40 mg/ml)]. The plate is placed in the incubator at 30 °C for 8 h to detect the LacZ reporter gene activity.
GST pull-down assay
APIP12F, APIP12N and APIP12M fragments were cloned into pGex-6p-1 in frame with GST tag to generate GST-APIP12F, GST-APIP12N and GST-APIP12M constructs. The mature form of AvrPiz-t (Avrpiz-t19-108) and APIP6 were cloned into pMAL-c2 vector in frame with MBP to generate MBP-AvrPiz-t and MBP-APIP6 constructs. Details of primers are given in Additional file 5: Table S1. Both GST-tagged and MBP-tagged vectors were transformed and expressed in E. coli strain Rosetta2 (DE3). The GST pull-down assay was conducted by following the procedure described previously (Park et al. 2016). About 2 μg of MBP- and GST-fused proteins were incubated at 25 °C for 2 h. The protein mixture was then incubated with 50 μg of glutathione-agarose beads for 2 h. Then the mixture was washed six times each with 1 ml of ice-cold PBS buffer. The eluted proteins after pull-down procedure were resolved in 10% SDS-PAGE gel and proteins were then transferred to Immobilon-PSQ PVDF membrane (Millipore). Diluted anti-GST antibody (Roche, USA) and anti-MBP antibody (Biomol, USA) were used for immunoblotting analysis. Chemiluminescene was detected using Pierce ECL substrate (Promega, USA) and the ChemiDoc XRS system (Bio-Rad).
Gene transcription analysis
Total RNAs were extracted from rice tissues using Trizol reagent by following the manufacturer’s instruction (Invitrogen, USA) and were then treated with DNase RQ1 (Promega, USA) to eliminate the contamination of genomic DNAs. Approximately 2 ug of total RNAs were used for reverse transcription using the Reverse Transcription System (Promega, USA). Semi-quantitative PCR was conducted with the procedure as described previously (Zhang et al. 2015a) to quantify the gene expression of APIP12 at different time points after rice blast and mock infection. The rice actin gene was used as a control in semi-quantitative PCR. Quantitative PCR was carried out using SYBR Green Supermix (TaKaRa, Japan) on a CFX96 96-well real-time PCR unit (Bio-Rad). The CFX96 software was used to calculate threshold cycle values. The data was normalized using the ubiquitin gene (OsUG) as an endogenous control and analyzed to calculate relative expression values using 2−ΔΔCt method (Livak and Schmittgen 2001). The two-tailed Student’s T-Test was used to determine the statistical significance. All the primers are listed in Additional file 7: Table S2. The expression analyses were repeated three times.
We described that the rice APIP12 protein was one of the host targets of the Magnaporthe oryzae avirulence effector AvrPiz-t and showed significant sequence similarity to Nup98 likely involved in the assembly of NPC. Functional characterization of APIP12 revealed that it was required for the basal resistance against rice blast. However, it was likely dispensable for the Piz-t mediated resistance.
AvrPiz-t interacting proteins
Green fluorescent proteins
Hours after infection
Nucleotide binding site
Pathogen-associated molecular patterns
Pattern recognition receptors
We gratefully acknowledge the courtesy of Dr. Changyin Wu at Huazhong Agriculture University, China for providing the Tos17 insertion line of the APIP12 gene in ZH11, TosRs_04Z11BB04. This study is supported by the Major Projects Transgenic Program of the Ministry of Agriculture (Grant No: 2012ZX08009001). This project is also supported by the grant from Natural Science Foundation in China (30971878) to B. Z. and by the Opening Project of State Key Laboratory of Crop Genetics & Germplasm Enhancement, Nanjing Agricultural University (ZW2014002).
The authors declare that they have no competing interests.
This study was conceived by BZ and GW. MT, YN, BD, HZ, XS, DW, and HW performed the experiments and data analysis. MT and BZ prepared the manuscript. All authors approved the manuscript.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Boeglin M, Fuglsang AT, Luu DT, Sentenac H, Gaillard I, Cherel I (2016) Reduced expression of AtNUP62 nucleoporin gene affects auxin response in Arabidopsis. BMC Plant Biol 16:2View ArticlePubMedPubMed CentralGoogle Scholar
- Bonardi V, Dangl JL (2012) How complex are intracellular immune receptor signaling complexes? Front Plant Sci 3:237View ArticlePubMedPubMed CentralGoogle Scholar
- Bos JI, Armstrong MR, Gilroy EM, Boevink PC, Hein I, Taylor RM, Zhendong T, Engelhardt S, Vetukuri RR, Harrower B, Dixelius C, Bryan G, Sadanandom A, Whisson SC, Kamoun S, Birch PR (2010) Phytophthora infestans effector AVR3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1. Proc Natl Acad Sci U S A 107:9909–9914View ArticlePubMedPubMed CentralGoogle Scholar
- Cesari S, Thilliez G, Ribot C, Chalvon V, Michel C, Jauneau A, Rivas S, Alaux L, Kanzaki H, Okuyama Y, Morel JB, Fournier E, Tharreau D, Terauchi R, Kroj T (2013) The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding. Plant Cell 25:1463–1481View ArticlePubMedPubMed CentralGoogle Scholar
- Chisholm ST, Coaker G, Day B, Staskawicz BJ (2006) Host-microbe interactions: shaping the evolution of the plant immune response. Cell 124:803–814View ArticlePubMedGoogle Scholar
- Dangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection. Nature 411:826–833View ArticlePubMedGoogle Scholar
- Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J, Foster GD (2012) The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13:414–430View ArticlePubMedGoogle Scholar
- Dodds PN, Rathjen JP (2010) Plant immunity: towards an integrated view of plant-pathogen interactions. Nat Rev Genet 11:539–548View ArticlePubMedGoogle Scholar
- Dou D, Zhou JM (2012) Phytopathogen effectors subverting host immunity: different foes, similar battleground. Cell Host Microbe 12:484–495View ArticlePubMedGoogle Scholar
- Du Z, Zhou X, Li L, Su Z (2009) plantsUPS: a database of plants’ Ubiquitin Proteasome System. BMC Genomics 10:227View ArticlePubMedPubMed CentralGoogle Scholar
- Du Y, Mpina MH, Birch PR, Bouwmeester K, Govers F (2015) Phytophthora infestans RXLR Effector AVR1 Interacts with Exocyst Component Sec5 to Manipulate Plant Immunity. Plant Physiol 169:1975–1990PubMedPubMed CentralGoogle Scholar
- Gohre V, Robatzek S (2008) Breaking the barriers: microbial effector molecules subvert plant immunity. Annu Rev Phytopathol 46:189–215View ArticlePubMedGoogle Scholar
- Iwamoto M, Asakawa H, Hiraoka Y, Haraguchi T (2010) Nucleoporin Nup98: a gatekeeper in the eukaryotic kingdoms. Genes Cells 15:661–669View ArticlePubMedGoogle Scholar
- Jones JD, Dangl JL (2006) The plant immune system. Nature 444:323–329View ArticlePubMedGoogle Scholar
- Kanzaki H, Yoshida K, Saitoh H, Fujisaki K, Hirabuchi A, Alaux L, Fournier E, Tharreau D, Terauchi R (2012) Arms race co-evolution of Magnaporthe oryzae AVR-Pik and rice Pik genes driven by their physical interactions. Plant J 72:894–907View ArticlePubMedGoogle Scholar
- Leung H, Raghavan C, Zhou B, Oliva R, Choi IR, Lacorte V, Jubay ML, Cruz CV, Gregorio G, Singh RK, Ulat VJ, Borja FN, Mauleon R, Alexandrov NN, McNally KL, Sackville Hamilton R (2015) Allele mining and enhanced genetic recombination for rice breeding. Rice (N Y) 8:34Google Scholar
- Li W, Wang B, Wu J, Lu G, Hu Y, Zhang X, Zhang Z, Zhao Q, Feng Q, Zhang H, Wang Z, Wang G, Han B, Wang Z, Zhou B (2009) The Magnaporthe oryzae avirulence gene AvrPiz-t encodes a predicted secreted protein that triggers the immunity in rice mediated by the blast resistance gene Piz-t. Mol Plant Microbe Interact 22:411–420View ArticlePubMedGoogle Scholar
- Liu W, Liu J, Ning Y, Ding B, Wang X, Wang Z, Wang G-L (2013) Recent progress in understanding PAMP-and effector-triggered immunity against the rice blast fungus Magnaporthe oryzae. Mol Plant 3:605–620View ArticleGoogle Scholar
- Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408View ArticlePubMedGoogle Scholar
- Miki D, Shimamoto K (2004) Simple RNAi vectors for stable and transient suppression of gene function in rice. Plant Cell Physiol 45:490–495View ArticlePubMedGoogle Scholar
- Ono E, Wong HL, Kawasaki T, Hasegawa M, Kodama O, Shimamoto K (2001) Essential role of the small GTPase Rac in disease resistance of rice. Proc Natl Acad Sci U S A 98:759–764View ArticlePubMedPubMed CentralGoogle Scholar
- Park CH, Chen S, Shirsekar G, Zhou B, Khang CH, Songkumarn P, Afzal AJ, Ning Y, Wang R, Bellizzi M (2012) The Magnaporthe oryzae Effector AvrPiz-t Targets the RING E3 Ubiquitin Ligase APIP6 to Suppress Pathogen-Associated Molecular Pattern–Triggered Immunity in Rice. Plant Cell 24:4748–4762View ArticlePubMedPubMed CentralGoogle Scholar
- Park CH, Shirsekar G, Bellizzi M, Chen S, Songkumarn P, Xie X, Shi X, Ning Y, Zhou B, Suttiviriya P, Wang M, Umemura K, Wang GL (2016) The E3 Ligase APIP10 connects the effector AvrPiz-t to the NLR receptor Piz-t in rice. PLoS Pathog 12:e1005529View ArticlePubMedPubMed CentralGoogle Scholar
- Qu S, Liu G, Zhou B, Bellizzi M, Zeng L, Dai L, Han B, Wang GL (2006) The broad-spectrum blast resistance gene Pi9 encodes a nucleotide-binding site-leucine-rich repeat protein and is a member of a multigene family in rice. Genetics 172:1901–1914View ArticlePubMedPubMed CentralGoogle Scholar
- Ratner GA, Hodel AE, Powers MA (2007) Molecular determinants of binding between Gly-Leu-Phe-Gly nucleoporins and the nuclear pore complex. J Biol Chem 282:33968–33976View ArticlePubMedGoogle Scholar
- Robles LM, Deslauriers SD, Alvarez AA, Larsen PB (2012) A loss-of-function mutation in the nucleoporin AtNUP160 indicates that normal auxin signalling is required for a proper ethylene response in Arabidopsis. J Exp Bot 63:2231–2241View ArticlePubMedPubMed CentralGoogle Scholar
- Schmidt HB, Gorlich D (2015) Nup98 FG domains from diverse species spontaneously phase-separate into particles with nuclear pore-like permselectivity. Elif 4:e04251Google Scholar
- Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599View ArticlePubMedGoogle Scholar
- van der Hoorn RA, Kamoun S (2008) From Guard to Decoy: a new model for perception of plant pathogen effectors. Plant Cell 20:2009–2017View ArticlePubMedPubMed CentralGoogle Scholar
- Wang R, Ning Y, Shi X, He F, Zhang C, Fan J, Jiang N, Zhang Y, Zhang T, Hu Y, Bellizzi M, Wang GL (2016) Immunity to rice blast disease by suppression of effector-triggered necrosis. Curr Biol 26:2399–2411View ArticlePubMedGoogle Scholar
- Wiermer M, Cheng YT, Imkampe J, Li M, Wang D, Lipka V, Li X (2012) Putative members of the Arabidopsis Nup107-160 nuclear pore sub-complex contribute to pathogen defense. Plant J 70:796–808View ArticlePubMedGoogle Scholar
- Wilton M, Subramaniam R, Elmore J, Felsensteiner C, Coaker G, Desveaux D (2010) The type III effector HopF2Pto targets Arabidopsis RIN4 protein to promote Pseudomonas syringae virulence. Proc Natl Acad Sci U S A 107:2349–2354View ArticlePubMedPubMed CentralGoogle Scholar
- Wu J, Kou Y, Bao J, Li Y, Tang M, Zhu X, Ponaya A, Xiao G, Li J, Li C, Song MY, Cumagun CJ, Deng Q, Lu G, Jeon JS, Naqvi NI, Zhou B (2015) Comparative genomics identifies the Magnaporthe oryzae avirulence effector AvrPi9 that triggers Pi9-mediated blast resistance in rice. New Phytol 206:1463–1475View ArticlePubMedGoogle Scholar
- Xu S, Powers MA (2013) In vivo analysis of human nucleoporin repeat domain interactions. Mol Biol Cell 24:1222–1231View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang Y, Li X (2005) A putative nucleoporin 96 is required for both basal defense and constitutive resistance responses mediated by suppressor of npr1-1, constitutive 1. Plant Cell 17:1306–1316View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang D, Liu M, Tang M, Dong B, Wu D, Zhang Z, Zhou B (2015a) Repression of microRNA biogenesis by silencing of OsDCL1 activates the basal resistance to Magnaporthe oryzae in rice. Plant Sci 237:24–32View ArticlePubMedGoogle Scholar
- Zhang S, Wang L, Wu W, He L, Yang X, Pan Q (2015b) Function and evolution of Magnaporthe oryzae avirulence gene AvrPib responding to the rice blast resistance gene Pib. Sci Rep 5:11642View ArticlePubMedGoogle Scholar