The Multivesicular Bodies (MVBs)-Localized AAA ATPase LRD6-6 Inhibits Immunity and Cell Death Likely through Regulating MVBs-Mediated Vesicular Trafficking in Rice

Previous studies have shown that multivesicular bodies (MVBs)/endosomes-mediated vesicular trafficking may play key roles in plant immunity and cell death. However, the molecular regulation is poorly understood in rice. Here we report the identification and characterization of a MVBs-localized AAA ATPase LRD6-6 in rice. Disruption of LRD6-6 leads to enhanced immunity and cell death in rice. The ATPase activity and homo-dimerization of LRD6-6 is essential for its regulation on plant immunity and cell death. An ATPase inactive mutation (LRD6-6E315Q) leads to dominant-negative inhibition in plants. The LRD6-6 protein co-localizes with the MVBs marker protein RabF1/ARA6 and interacts with ESCRT-III components OsSNF7 and OsVPS2. Further analysis reveals that LRD6-6 is required for MVBs-mediated vesicular trafficking and inhibits the biosynthesis of antimicrobial compounds. Collectively, our study shows that the AAA ATPase LRD6-6 inhibits plant immunity and cell death most likely through modulating MVBs-mediated vesicular trafficking in rice.


Introduction
Plants are exposed to a vast diversity of micro-organisms such as bacteria, fungi and oomycetes. To protect themselves from pathogenic plant-microbe interactions, plants have developed a sophisticated innate immunity system [1]. Pattern triggered immunity (PTI) and effector triggered immunity (ETI) are two major layers of an immunity system that shares many common responses to pathogen infection including protein phosphorylation, hormonal change, ion fluxes change, production of reactive oxygen species (ROS), synthesis of antimicrobial compounds, transcriptional activation of pathogenesis-related (PR) genes and cell-wall reinforcement via oxidative cross-linking of cell-wall components and deposition of lignin [2][3][4]. Cell death, which plays a central role in many plant processes, has been observed in both PTI and ETI [3,5,6]. Upon perception of pathogens, the immunity system activates rapid cell death, characterized as a form of hypersensitive response (HR) typically in and around the infection sites to restrict pathogen invasion and prevent disease development [7,8].
Previous studies have shown that protein trafficking mediated by multivesicular bodies (MVBs) is associated with immunity in plants [31,32]. Upon perception of ligand flagellin To determine if cell death occurred in the lrd6-6 mutant, we stained rice leaves using trypan blue. The blue staining spots were present in 8-d-old lrd6-6 plants even before initiation of lesion spots, and more blue staining spots accumulated along with plant development (Fig 1B, upper panel). However, no blue staining spots appeared in leaves of Kitaake ( Fig 1B, upper  panel). Further, DAB (3, 3'-diamiobenzidine) analysis detected extensive stains in leaves of lrd6-6 before lesion spots appearance. On the contrary, almost no staining occurred in Kitaake along with plant development (Fig 1B, lower panel), indicating that excess hydrogen peroxide had accumulated in the lrd6-6 mutant compared with Kitaake. We then sectioned the leaves of lrd6-6 and Kitaake, and observed under transmission electron microscopy. The subcellular structures were severely degraded in the lesion-spotted parts of leaves but not in other leaf parts in absence of lesion spots in lrd6-6 or the equivalent parts of Kitaake (S4 Fig). These results suggest that spontaneous cell death occurs in the lrd6-6 mutant, which results in the formation of lesion spots.
Previous studies have shown that cell death in plants is usually mediated by enhanced immunity [12,47,48]. We therefore determined the expression levels of immunity related genes, such as the PR genes, Betv1, OsNPR10, OsPR1a and 04g10010 [30,49,50]. The expression of these PR genes all increased in lrd6-6 compared with Kitaake plants, predominantly after the presence of cell death ( Fig 1C). We then challenged lrd6-6 plants with the fungal pathogen Magnaporthe oryzae (M. oryzae) and the bacterial pathogen Xanthomonas oryzae pv oryzae (Xoo), which cause blast and bacterial blight diseases, respectively. The disease lesion length and the number of lesions on leaves inoculated with M. oryzae strains (ZB25, Zhong1 and ZE-1) compatible with Kitaake, were all dramatically reduced in the lrd6-6 mutant compared with Kitaake ( Fig 1D). When inoculated with compatible Xoo strains (P2, P4, P5, P6 and Xoo-4), the lrd6-6 mutant also exhibited enhanced resistance, showing much shorter disease lesion length than Kitaake plants (Figs 1E and S5). Collectively, our results indicate that the rice lrd6-6 mutant possesses enhanced immunity, which may be mediated by the cell death in the lrd6-6 mutant.

Lrd6-6 encodes a putative AAA ATPase
Since the lrd phenotype caused by cell death in lrd6-6 did not co-segregate with the hygromycin (Hyg) gene (S6 Fig), we presumed that the spontaneous cell death in the lrd6-6 mutant might have resulted from tissue-culture induced somatic mutation during transformation. We thus developed three F 2 populations and performed genetic analysis on the lrd6-6 locus. Genetic and after (AL) lesion spots appeared on the lrd6-6 mutant. The expression levels of the PR genes were normalized to the Ubq5 reference gene. Error bars represent the standard deviations (SDs) for three biology repeats and the expression differences was determined by Student's t-test (**, P < = 0.01). (D) Determination of the resistance of the lrd6-6 mutant to blast disease. Photographs of representative leaves were respectively taken at 7 d post-inoculation with M. oryzae strains, ZB25, Zhong-1 and ZE-1. Statistical analyses of the disease lesion lengths and lesion numbers were respectively performed on the leaves of inoculated Kitaake and lrd6-6 (error bar, SEM, n > 30). Asterisks denote a significant difference from the wild type as determined by Student's t-test (**, P < = 0.01). (E) Determination of the resistance of the lrd6-6 to bacterial blight disease. Photographs of representative leaves were taken at 15 d postinoculation (DPI) with Xoo strain P2, which is compatible with Kitaake (a). Disease lesion lengths (b) and bacterial populations (c) of the lrd6-6 mutant and Kitaake were measured at 0, 7 and 14 DPI (error bar, SEM; n > 10 for lesion lengths and n = 5 for bacterial populations). Asterisks denote a significant difference from the wild type as determined by Student's t-test (*, P < = 0.05; **, P < = 0.01).
doi:10.1371/journal.pgen. 1006311.g001 analyses showed that the lrd6-6 phenotype was controlled by a single recessive nuclear locus (S1 Table). Next, map-based cloning of the Lrd6-6 gene was performed using 344 F 2 individuals with the spontaneous cell death phenotype from the cross between the lrd6-6 mutant and rice 02428. The Lrd6-6 locus was first mapped in the interval with a physical distance of 2.54 Mb between the InDel markers I4-2 and I8 on chromosome 6 (Fig 2Aa). By using more markers to analyze 2175 F 2 individuals with the cell death phenotype, the Lrd6-6 locus was then delimited on the genomic region within 93 kb between RM8075 and RM587 (Fig 2Aa). Next, we sequenced the genomic DNA sample bulked with 30 BC 2 F 3 individuals with cell death phenotypes using a whole-genome resequencing approach. The genomic DNA of Kitaake was also sequenced as a control. When comparing the sequences of the 93 kb between the bulked DNA sample and the Kitaake control DNA, we found that a 1446 bp DNA fragment from gene LOC_Os06g03940 (RGAP ID from http://rice.plantbiology.msu.edu, abbreviated to Os06g03940 hereafter) that spans four exons was tandemly repeated in Os06g03940 in the bulked DNA sample (Fig 2Ab). Sequencing of cDNA revealed that this 1446 bp tandem repeat resulted in an insertion of 534 bp in the protein-coding sequence of Os06g03940 that might disrupt the gene function in the lrd6-6 mutant (S7 Fig). These results indicate that the spontaneous cell death phenotype in the lrd6-6 mutant was likely resulted from the insertion of 1446 bp tandem repeat in the Os06g03940 gene, and thus Os06g03940 encoding an AAA ATPase is likely the target gene of Lrd6-6.
We carried out a knockdown experiment to confirm that the lrd6-6 mutation was caused by Os06g03940. We amplified a unique segment (Seg I, 445 bp) covering nucleotides 557 to 1001 of the Os06g03940 open reading frame (ORF) (S8 Fig) and used this segment to create an RNA interference (RNAi) construct, pANDA-Os06g03940Ri. This segment shows only approximately 20.12% identity with the closest homologous gene Os01g04814 (S8 Fig). This construct was then introduced into Kitaake through Agrobacterium-mediated transformation. We found that all 33 transgenic plants with suppressed Os06g03940 expression displayed spontaneous cell death similar to the lrd6-6 mutant (Figs 2B and S9A). In these plants, the expression of Os01g04814, which shared the highest identity with Os06g03940 in cDNA sequence, was not suppressed (Fig 2Bd), suggesting that specific silencing of Os06g03940 resulted in the spontaneous cell death phenotype.
To further verify the result obtained by RNAi analysis, we then cloned an 11.5 kb genomic DNA fragment harboring the native promoter and full coding region of the gene Os06g03940 from rice Nipponbare and placed it in the binary vector pCactN-XG to create the construct pCactN-XG-Os06g03940-11.5kb. The DNA fragment Os06g03940-11.5kb was then introduced into the lrd6-6 mutant through a similar Agrobacterium-mediated transformation approach. All 12 transgenic lines carrying the transgene Os06g03940-11.5kb no longer exhibited the spontaneous cell death phenotype (Figs 2C and S9B). This reveals that the Os06g03940-11.5kb transgene restores the spontaneous cell death phenotype in the lrd6-6 mutant to that of the wild type Kitaake. Taken together, these results clearly demonstrate that Os06g03940 is the target gene of lrd6-6, in which the mutation with the inserted 1446 bp tandem repeat was responsible for the spontaneous cell death phenotype.
Recently, Fekih et al. reported that a G->A base substitution resulted in a premature translation termination in the Lmr gene (RAP ID Os06g0130000; http://rapdb.dna.affrc.go.jp), which is the same gene Os06g03940 from RGAP according to gene ID conversion through ID Converter (http://rapdb.dna.affrc.go.jp/tools/converter), and also led to the spontaneous cell death phenotype in rice [51]. Similar to lrd6-6, the lmr mutant displayed elevated PR gene expression and enhanced disease resistance compared with wild type rice Hitomebore [51]. Fekih and his colleagues also used the RNAi approach targeting Seg II of the gene, which spans a region different from Seg I used in our study (S8 Fig), and a complementary test by Positional cloning of Lrd6-6. (A) Fine mapping of the Lrd6-6 locus. The Lrd6-6 locus was delimited to a 93 kb interval between SSR markers RM8075 and RM587 on chromosome 6 (a). The molecular markers and the number of recombinants are shown. A structure variation (SV) was identified from the target gene Os06g03940 in the lrd6-6 mutant. The 1446 bp tandem repeat was found in the gene Os06g03940 which resulted in an additional insertion of 534 bp in the coding DNA of Os06g03940 in lrd6-6 (b). The SV mutation was detected by a whole genome resequencing overexpressing the full-length cDNA of Lmr to confirm their results [51]. Together, these results confirm that disruption of Os06g03940 leads to the lrd6-6 phenotype.
Lrd6-6 is expressed in diverse tissues in rice and the protein resides mainly on MVBs To determine the expression pattern of Lrd6-6, we respectively sampled the root, stem, leaf and panicle at the two-, four-, six-leaf and mature stages of Kitaake and determined the transcript levels of Lrd6-6 in these tissues. The quantitative reverse transcription-PCR (qRT-PCR) analysis showed that Lrd6-6 was expressed in all these tissues with predominance in leaves (S10 Fig).
To investigate the subcellular localization of LRD6-6, we generated construct p35S-Lrd6-6-GFP expressing the LRD6-6-GFP fusion protein and performed a transient expression assay using Nicotiana benthamiana through an agroinfiltration approach. The GFP signal was punctate in the cytoplasm in the leaf of N. benthamiana expressing LRD6-6-GFP fusion protein whereas, as expected, the GFP fluorescence was distributed in the cytoplasm and nucleus in the leaf expressing GFP alone (S11A Fig). The distribution of the LRD6-6-GFP fusion protein was reminiscent of the localization of the Arabidopsis LRD6-6 homologous protein AAA ATPase SKD1, which was previously shown to be located in MVBs [38]. By using bombardment-mediated transformation, we also observed a punctate pattern of GFP signals in onion epidermal cells expressing the LRD6-6-GFP fusion protein (S11B Fig). The Lrd6-6-GFP transgene was able to restore the lrd6-6 plants to the wild type level (S12 Fig) suggesting that the LRD6-6-GFP fusion protein functions similarly as LRD6-6. The MVBs-localized pattern of the LRD6-6-GFP protein highly suggests that LRD6-6 localizes in MVBs.
The Rab GTPase RabF1/ARA6 protein has been shown to locate on the peripheral membrane of the MVBs and has been widely used as the specific marker protein for plant MVBslocalization analysis [52,53]. We transiently expressed the fused proteins, RabF1/ARA6-GFP and RabF1/ARA6-RFP respectively in N. benthamiana cells through the same agroinfiltration approach and found that both RabF1/ARA6-GFP and RabF1/ARA6-RFP are present in punctate patterns in the cells as expected (S13 Fig). When treated with chemical wortmannin which is able to change MVBs into ring-like structures through inhibiting phosphatidylinositol 3-kinase (PI3) activity [54,55], the punctate GFP or RFP signals in the N. benthamiana cells expressing RabF1/ARA6-GFP or RabF1/ARA6-RFP were respectively converted into ring-like structures (S13 Fig). These results suggested that RabF1/ARA6-GFP and RabF1/ARA6-RFP were MVBs-localized and could be used as control for MVBs-localization analysis on LRD6-6. Then, we co-transformed LRD6-6-RFP (RFP fused on the C-terminus of LRD6-6) with RabF1/ARA6-GFP, LRD6-6-GFP (GFP fused on the C-terminus of LRD6-6) or YFP-LRD6-6 approach and was confirmed by PCR-based sequencing. (B) Plants of RNAi-mediated silencing of Lrd6-6 exhibited spontaneous cell death phenotypes. The photographs of five representative transgenic T 0 lines with cell death (#1-#5) and the control (CK) Kitaake are shown (a). Representative leaves from the plants indicated in (a) are shown in (b). Transcriptional expression levels of Os06g03940 and LOC_Os01g04814 which shares the highest identity with Os06g03940 in cDNA sequence in rice plants were respectively determined (c) and (d). PCR-based genotyping with the primer pair specific for the hygromycin (Hyg) gene was performed to determine whether the plants contained (represented by '+') or lacked (represented by '-') the transgene Os06g03940Ri. The relative expressions of the genes were normalized to the Ubq5 reference gene. The error bars represent the SDs of three biology repeats and the expression differences was determined by Student's t-test (*, P < = 0.05; **, P < = 0.01; N.D., No significantly difference). (C) An 11.5 kb wild type genomic segment of Lrd6-6 completely rescued the cell death phenotype of the lrd6-6 mutant. Photograph of three independent representative T 0 plants carrying the genomic DNA fragment of Lrd6-6 (Os06g03940-11.5kb) in the lrd6-6 mutant genetic background (a)-Kitaake and the lrd6-6 mutant are also included in the photograph. PCR-based genotyping of the Neomycin phosphotransferase II (NPT II) gene indicated that the plants contained ('+') or lacked ('-') the transgenic genome DNA fragment Os06g03940-11.5kb. Representative leaves from the plants indicated in (a) are shown in (b). Bars = 10 cm. (YFP fused on the N-terminus of LRD6-6), respectively, with RabF1/ARA6-RFP. Consistently with prior results, the punctate fluorescence signals of the fusion proteins, LRD6-6-GFP, LRD6-6-RFP and YFP-LRD6-6, all co-located with fluorescence signals derived from RabF1/ ARA6-GFP or RabF1/ARA6-RFP protein but not the signals of chlorophyll (Figs 3 and S14). Together, our results clearly show that the LRD6-6 protein resides mainly on MVBs which is different from the results of Fekih et al. showing that LRD6-6-GFP was localized in chloroplast [51].

LRD6-6 can homo-dimerize in yeast and in planta
Previous reports have shown that the AAA ATPase VPS4/SKD1, which is homologous to LRD6-6, can form dimers to function in disassembly of the ESCRT-III complex in regulation of MVBs biogenesis [37,58,60]. To test if LRD6-6 possesses the ability of dimerization, we cloned the full-length coding sequences (CDSs) of Lrd6-6, Lrd6-6 K261A , Lrd6-6 E315Q and Lrd6-6 R372E respectively into both pGADT7 and pGBKT7 vectors and used for yeast two hybrid (Y2H) analysis. The CDS of lrd6-6 (Lrd6-6 m ) was also cloned into these vectors and included in this analysis. The results showed that the protein LRD6-6 interacted with itself ( Fig 5A). Variants LRD6-6 K261A and LRD6-6 E315Q also interacted with themselves whereas variants LRD6-6 R372E and LRD6-6 m did not ( Fig 5A). We then fused these proteins with the split YFP N-half and split YFP C-half, respectively, and performed bimolecular fluorescence complementation (BiFC) assay in N. benthamiana. The results showed that the cells of N. benthamiana co-transformed with LRD6-6-YFP N and LRD6-6-YFP C , LRD6-6 K261A -YFP N and LRD6-6 K261A -YFP C , or LRD6-6 E315Q -YFP N and LRD6-6 E315Q -YFP C produced yellow fluorescence signals whereas the cells co-transformed with LRD6-6 m -YFP N and LRD6-6 m -YFP C , LRD6-6-YFP N and LRD6-6 m -YFP C , or LRD6-6 R372E -YFP N and LRD6-6 R372E -YFP C , exhibited no fluorescence signals (Fig 5B). These results suggest that LRD6-6 is capable of homo-dimerization and-of the three residues, K261, E315 and R372 -only R372 was required for this dimerization. As residues K261 and E315 are essential for ATPase activity but not required for homo-dimerization, these results also indicate that the ATPase activity is not required for homo-dimerization of LRD6-6. However, residue R372 is required for both homo-dimerization and ATPase activity of LRD6-6, suggesting that homo-dimerization may be required for the ATPase activity of LRD6-6.

ATPase activity is required for full function of LRD6-6 in rice
To determine if the ATPase activity of LRD6-6 is required for its biological function, we created constructs, which carry Lrd6-6 K261A , Lrd6-6 E315Q or Lrd6-6 R372E with the ATPase activity knocked out or compromised, and introduced them individually into the lrd6-6 mutant. Transformation of Lrd6-6 into lrd6-6 was also performed as a positive control. The transgenic plants expressing the wild type LRD6-6 were able to restore the lrd6-6 phenotype to wild type (S16 Fig); however, none of LRD6-6 K261A , LRD6-6 R372E and LRD6-6 E315Q restored the mutant ( Fig  6). The lrd6-6 plants expressing the LRD6-6 with catalytically inactive or compromised ATPase activity retained enhanced immunity and spontaneous cell death as the lrd6-6 mutant (Fig 6). Thus, the ATPase activity is essential for LRD6-6 to inhibit immunity and cell death in rice.
Expression of the catalytically inactive mutant Lrd6-6 E315Q leads to autoimmunity and spontaneous cell death in rice The glutamate (E) of hhhhDE sequence (h represents a hydrophobic amino acid) in the Walker B motif is crucial for ATP hydrolysis while the lysine (K) residue in the Walker A consensus sequence GXXXXGK[T/S] (X is any amino acid) of the conserved AAA ATPase family is crucial for ATP binding [36]. Although these two residues are capable of rendering the wild type protein dominant-negative [36], mutation of E in the Walker B motif has been more widely used to create 'substrate traps' in yeast [37], mammals [61] and Arabidopsis [38]. Because LRD6-6 E315Q could dimerize with LRD6-6 as shown in yeast and in N. benthamiana (Fig 7A), we presumed that LRD6-6 E315Q would also play a dominant-negative role for LRD6-6 in rice.  E315Q and His-LRD6-6(125-487) R372E . ATPase activities were measured using a malachite green-based colorimetric approach. The ATPase activities with mean values ± SEM of four replications were shown. Statistical significance comparison was conducted with ANOVA (P < = 0.01), where different capital letters above columns indicate significant differences, whereas the same letter indicates no significant differences.  The full-length CDSs for LRD6-6, the mutant lrd6-6 (LRD6-6 m ) and the variants, LRD6-6 K261A , LRD6-6 E315Q and LRD6-6 R372E were respectively inserted into the pGPBKT7 (BD) and pGADT7 (AD), and were subjected to Y2H test through co-transformation by pairs (left panel). The pairs, pGBKT7-53/pGADT7-T and pGBKT7-Lam/pGADT7-T, representing positive and negative interactions, respectively, were also included in this test. The positive transformants were diluted in 10-fold serial dilution and spotted on double dropout medium To test this hypothesis, we expressed Lrd6-6 E315Q in wild type Kitaake. The result showed that the transgenic plants expressing LRD6-6 E315Q displayed spontaneous cell death similar to the lrd6-6 mutant plants, while transgenic plants expressing the wild type LRD6-6 did not (Figs 7B and S17). Detection of dead cells using trypan blue and measurement on the expression of PR genes indicated that the transgenic plants expressing LRD6-6 E315Q also possessed enhanced immunity and presented spontaneous cell death similarly as the lrd6-6 mutant ( Fig 7C). These results show that the LRD6-6 E315Q is able to compromise LRD6-6 function likely by forming a functionally inactive homo-dimer, thus acting dominant-negatively. These results also suggest that mutant gene Lrd6-6 E315Q may be utilized as a gene trap to suppress the inhibitory regulation of the ATPase LRD6-6 in immunity and cell death to enhance plant disease resistance.
Genes associated with MVBs-mediated vesicular trafficking are dysregulated in the lrd6-6 mutant To identify downstream components of the immunity and cell death resulting from disruption of the LRD6-6 ATPase in lrd6-6, we performed a genome-wide transcript comparative analysis on lrd6-6 and Kitaake using an RNA-seq approach. A total of 1223 differentially expressed genes (DEGs) were obtained (S18 Fig). Of these, 980 genes were up-regulated whereas 243 were down-regulated in the lrd6-6 mutant compared to the wild type Kitaake [P < = 0.05, Log 2 FC (lrd6-6/Kitaake) > 1] (S18 Fig).
Gene Ontology (GO) analysis showed that these DEGs could be classified into different cellular components (S19 Fig and S2 Table). Of them, MVBs-mediated vesicular trafficking associated components were the most enriched, including the GO terms membrane, membrane coat, clathrin coat of coated pit, endoplasmic reticulum membrane and clathrin coat of trans-Golgi network vesicle (S19 Fig and S2 Table). We then randomly selected some of these genes, performed qRT-PCR analysis on them and verified their differential expression between lrd6-6 and Kitaake (S20 Fig). We also investigated the expression of these genes in lrd6-6 plants transformed with the Os06g03940-11.5kb transgene and plants expressing Lrd6-6 E315Q by qRT-PCR analysis. The results showed that expression of Os06g03940-11.5kb in lrd6-6 restored the expressions of these genes to the similar levels in the wild type Kitaake plants while expression of Lrd6-6 E315Q in Kitaake retained dysregulation of these genes similarly as lrd6-6 (S21 Fig). These results suggested that the dysregulation of these genes in lrd6-6 is indeed the result of loss of function of the Os06g03940 (Lrd6-6) gene. Since a crosstalk exists between the secretory pathway and the early endocytic route in the early MVBs/trans-Golgi network [62], we therefore tested whether some genes associated with both secretory and endocytic trafficking were influenced by dysfunction of the AAA ATPase LRD6-6. Indeed, the expression of gene Os01g74180, encoding the β-subunit of adaptor protein complex 3 (AP-3), whose homologs have been shown to be important regulators of both endocytic and secretory pathways in yeast, mammals and Arabidopsis [63][64][65], was down-regulated in lrd6-6 plants and the Kitaake plants carrying Lrd6-6 E315Q (S20 and S21 Figs, S2 Table). The clathrin heavy chain gene (chcA) was (SD/-Leu/-Trp; DDO) and quadruple dropout medium supplemented with Aureobasidin A (SD/-Ade/-His/-Leu/-Trp/AbA; QDO/ A) plates. The growth of co-transformed yeast cells in the medium QDO/A indicates protein-protein interaction in yeast. Western blot analysis with anti-c-myc or anti-HA antibodies was performed to ensure the expression of BD-and AD-fused proteins, respectively, for each co-transformation event (right panel). (B) BiFC analyses on the dimerization of LRD6-6 in N. benthamiana. The full-length CDSs for LRD6-6, the mutant lrd6-6 (LRD6-6 m ) and the variants, LRD6-6 K261A , LRD6-6 E315Q and LRD6-6 R372E were respectively inserted into the split yellow fluorescence protein (YFP) vectors, split YFP N and split YFP C , and were cotransformed into N. benthamiana cell by pair. The punctate YFP signals represent interaction between the co-expressed proteins, while the red signals represent the auto-fluorescence of chlorophyll. The YFP signals didn't overlap with the auto-fluorescence, bars = 20 μm. The expression of proteins was detected by anti-c-myc or anti-HA respectively. or Lrd6-6 R372E (C) in the lrd6-6 mutant genetic background was not able to rescue the autoimmunity and cell death phenotype of lrd6-6. For each transformation, leaves (a) from four independent lines with gene expression verified (b) are shown. Of them, two lines were subjected to cell death (c) and PR genes expression analyses (d), respectively. The relative expressions of the genes were normalized to the Ubq5 reference gene. The error bars represent the SDs of three biology repeats and the expression differences was determined by Student's t-test (*, P < = 0.05; **, P < = 0.01).  reported to be essential for secretion of lysosomal enzymes in Dictyostelium discoideum [66]. In Arabidopsis, the chc2 single mutant and dominant-negative CHC1 (HUB) transgenic lines were defective in bulk endocytosis and in internalization of prominent plasma membrane proteins [67]. We also found the Os12g01390 gene, coding for the clathrin heavy chain was up-regulated in lrd6-6 based on the RNA-seq data (S2 Table). These results indicate that MVBsmediated vesicular trafficking is associated with the ATPase LRD6-6 and this trafficking process is dysregulated in lrd6-6 and the Kitaake plants expressing Lrd6-6 E315Q .
Previous studies have shown that the Arabidopsis soluble vacuolar Carboxypeptidase Y (AtCPY) is transported from endoplasmic reticulum (ER) to the vacuole through the early secretory pathway mediated by MVBs [68]. Dysregulation of MVBs-mediated vesicular trafficking inhibits the AtCPY-GFP transport in Arabidopsis [45]. To detect whether the MVBsmediated vesicular trafficking process was dysregulated in lrd6-6, we transiently expressed AtCPY fused to the N-terminal GFP (AtCPY-GFP) in protoplasts prepared from Kitaake and lrd6-6, respectively. The GFP fluorescence signal of AtCPY-GFP in Kitaake protoplasts could be visualized in the vacuole (Fig 8Aa and 8C). By contrast, the fluorescence was prominently presented in cytoplasm in the lrd6-6 protoplasts (Fig 8Ab and 8C), showing that the trafficking of AtCPY-GFP from ER to the vacuole was obviously inhibited in lrd6-6. The transport of AtCPY-GFP in the lrd6-6 plants expressing Os06g03940-11.5kb transgene was restored to the normal level of the wild type Kitaake plants whereas inhibited in the Kitaake plants expressing Lrd6-6 E315Q (Fig 8Ac, 8Ad and 8C). These results clearly showed that the MVBs-mediated vesicular trafficking was modulated by the AAA ATPase LRD6-6. To determine whether dysfunction of MVBs could inhibit the transport of AtCPY-GFP in rice, we treated the Kitaake protoplasts transiently expressing AtCPY-GFP with wortmannin. We found that the transport of AtCPY-GFP was inhibited in most protoplasts (about 80%) treated with wortmannin ( Fig  8B and 8C). This result clearly shows that transport of rice-expressed AtCPY-GFP from ER to vacuoles is also mediated by MVBs and this transport is completely inhibited in the lrd6-6 mutant.
Taken together, our results suggest that the MVBs-mediated vesicular trafficking is altered in the lrd6-6 mutant and the autoimmunity and spontaneous cell death of lrd6-6 likely results from the dysregulated MVBs-mediated vesicular trafficking.
To determine whether loss-of-function of LRD6-6 affects MVBs in general, we transiently expressed RabF1/ARA6-GFP in the protoplast cells prepared from Kitaake and lrd6-6 respectively. The result showed that localization of RabF1/ARA6-GFP in lrd6-6 was not changed compared to that observed in Kitaake (S22 Fig). This result suggested that LRD6-6 does not affect the machinery of MVBs generally.

Antimicrobial metabolites accumulate in the lrd6-6 mutant
Among the DEGs identified with RNA-seq, many were associated with immunity and cell death according to GO biological function analysis. These included PR genes, chitinase, WRKY transcription factors, MPKs and oxidation-related genes (S3 Table). We randomly selected some of these genes and verified their differential expression between the lrd6-6 mutant and wild type Kitaake (S23 Fig).
To explore the downstream events involved in the autoimmunity and spontaneous cell death in the lrd6-6 mutant, we performed pathway analysis on the DEGs-many pathways were likely involved in the immunity and cell death (S4 Table). Of them, three pathways, phenylpropanoid biosynthesis, diterpenoid biosynthesis and phenylalanine, tyrosine and tryptophan biosynthesis, which contribute to innate immunity and cell death according to previous reports  [69][70][71] were highly activated in the lrd6-6 mutant compared with the wild type Kitaake (S24 Fig and S4 Table).
Gene pathway analysis also showed that the antimicrobial metabolites, including serotonin, lignin and phytoalexins might accumulate in the lrd6-6 mutant (Figs 9A and 10A). We thus performed qRT-PCR to analyze the expression of the key enzyme genes involved in biosynthesis of these antimicrobial metabolites. Indeed, we found that expression of the genes aroA and aroC, required for biosynthesis of chorismate from shikimate [72,73], obviously increased in the lrd6-6 mutant (Fig 9A). Because chorismate is required for biosynthesis of both serotonin and lignin [74,75], the increased expression of aroA and aroC would lead to accumulation of The key enzyme genes with increased expression in the lrd6-6 mutant detected by RNA sequencing are shown in red. Data in the brackets are the gene expression fold-changed determined by qRT-PCR with gene specific primers (Fold-changed ± SD), asterisks denote statistical gene expression increases in lrd6-6 mutant compared to Kitaake by Student's t-test (*, P < = 0.05; **, P < = 0.01). (B) Comparison of the expression of OsWRKY14, which regulates synthesis of serotonin directly, between Kitaake and lrd6-6. (C) Content determination of lignin in Kitaake and lrd6-6. The bars represent the means of data from a representative experiment, and the error bars indicate the SEM (n = 4). Asterisks denote a significant difference between the lrd6-6 mutant and the wild type Kitaake (Student's t-test; **, P < 0.01).  the antimicrobial metabolites, serotonin and lignin. We then found that expression of the downstream genes essential for respective biosynthesis of serotonin and lignin initiated from chorismate (for biosynthesis of serotonin: As, Igps, Ts and Tdc; for biosynthesis of lignin: Pal, Ptal, 4Cl, Ccr, F5h, Cad and Pox) increased in the lrd6-6 mutant (Fig 9A), supporting our notion that the antimicrobial metabolites, serotonin and lignin, would accumulate in the lrd6-6 mutant. Then we measured the expression of the genes required for the biosynthesis of phytoalexins, including phytocassanes A-E (genes: OsCyc2/OsCps2 and OsDtc1/OsKsl7), oryzalexins A-F (genes: OsCyc2/OsCps2 and OsDtc1/OsKsl10), oryzalexin S (genes: OsCyc1/OsCps4 and OsDtc2/OsKsl8) and momilactones A and B (genes: OsCyc1/OsCps4 and OsDtc1/OsKsl4) [76] between the lrd6-6 mutant and Kitaake ( Fig 10A). These genes expressed higher in the lrd6-6 mutant compared with Kitaake ( Fig 10A). This result suggested that biosynthesis of these phytoalexins might be highly activated in the lrd6-6 mutant. Previous reports have shown that the WRKY transcription factor OsWRKY14 is required for serotonin biosynthesis through regulating the expression of genes TS (tryptophan synthase) and TDC (tryptophan decarboxylase) [74,77] and that the MPK genes OsMPK3, OsMPK6 and OsMPK4 regulate the biosynthesis of lignin and phytoalexins [75,78]. When determining the expression of these genes, with the exception of OsMPK6, we found that the expression of all these genes was highly increased in the lrd6-6 mutant compared with Kitaake (Figs 9B and 10B), further supporting our notion that antimicrobial metabolites accumulated in the lrd6-6 mutant.
To confirm that these antimicrobial metabolites accumulate in lrd6-6, we selectively measured and compared the contents of lignin and phytoalexins between lrd6-6 and Kitaake. The result showed that the amount of total lignin and phytoalexins, momilactones A and B, and phytocassanes A-E in lrd6-6 were indeed largely increased compared with Kitaake (Figs 9C and 10C). Consistently, the contents of phytoalexins in the lrd6-6 plants expressing the transgene Os06g03940-11.5kb was restored to the levels of the wild type Kitaake while highly accumulated in the transgenic Kitaake plants expressing Lrd6-6 E315Q which exhibited cell death (S25 Fig). Previous study has shown that salicylic acid (SA) biosynthesis is involved in shikimate-phenylpropanoid pathway [79], which is also involved in lignin biosynthesis activated in lrd6-6. Elevated levels of SA could stimulate SA-mediated defense response leading to secretion of antimicrobial compounds and increased expression of PR genes in plant [80]. To determine whether SA was accumulated in lrd6-6, we sampled the leaf of the lrd6-6 mutant, the lrd6-6 plant expressing the transgene Os06g03940-11.5kb, the Kitaake plant expressing Lrd6-6 E315Q and the wild type Kitaake and subjected them to total SA content determination. The result showed that there was no significant difference of SA contents among the samples (S26 Fig). Taken together, these results suggested that the enhanced immunity and cell death in lrd6-6 results from accumulation of phytoalexins. These results also indicate that the accumulation of the antimicrobial metabolites instead of SA in the lrd6-6 mutant likely directly inhibit the infection of pathogens such as M. oryzae and Xoo.

LRD6-6 interacts with ESCRT-III components OsSNF7 and OsVPS2
To further dissect the molecular mechanism of LRD6-6 in regulation of immunity and cell death, we performed an Y2H screen using LRD6-6 as bait. The full amino acid coding sequence between the lrd6-6 mutant and Kitaake. The expression levels of the genes were normalized to the Ubq5 reference gene. Error bars represent the SDs for three biology repeats and the expression differences was determined by Student's t-test (**, P < = 0.01; N.D., No significantly difference). (C) Content determination of the phytoalexins as indicated in Kitaake and lrd6-6. The data was obtained from four biological replicates. The error bars indicate the SEMs. Asterisks denote a significant difference between the lrd6-6 mutant and Kitaake (Student's t-test; **, P < 0.01).
doi:10.1371/journal.pgen.1006311.g010 of Lrd6-6 was cloned in-frame with the GAL4 DNA binding domain of the bait vector pGBKT7. The Y2H screen was conducted using a cDNA library derived from rice Nipponbare. We identified a SNF7 domain containing protein (Os06g40620) and named this protein OsSNF7 because it showed high identity with Arabidopsis SNF7 in amino acid sequence. To confirm its interaction with LRD6-6, we amplified the full-length CDS of OsSnf7 and cloned it into the prey vector to obtain pGADT7-OsSNF7. The Y2H test clearly showed that full-length protein of OsSNF7 interacted with LRD6-6 ( Fig 11A). We then fused OsSNF7 with GFP, LRD6-6 with RFP respectively and co-expressed them into N. benthamiana and observed their localizations. The result showed most of the green fluorescence produced by OsSNF7-GFP overlapped with the red fluorescence signals of LRD6-6-RFP (S27 Fig). This result suggests that the OsSNF7 protein also resides in MVBs and co-localizes with LRD6-6. The interaction between OsSNF7 and LRD6-6 was further confirmed by BiFC analysis in N. benthamiana through co-expressing LRD6-6-YFP N /OsSNF7-YFP C (Fig 11B).
To test if OsSNF7 functions in immunity and cell death in plants, we knocked out (KO) OsSnf7 in rice Kitaake using the CRISPR/CAS9 approach [81]. Three independent OsSnf7-KO rice lines with premature stop mutation at different residues were obtained (S28 Fig). Unexpectedly, these OsSnf7-KO plants, which were homozygous for their respective premature stop mutation, displayed no obvious cell death or growth defects compared with Kitaake. The expression of PR genes in these lines remained at the same level as wild type Kitaake (S29 Fig). These results suggested that the knock-out of OsSnf7 did not affect the immune response or cell death in plants. We then analyzed the homologs of OsSNF7 in rice and found four proteins encoded by genes Os12g02830, Os11g03060, Os09g09480 and Os07g30830 that also shared high identities with OsSNF7 ( S30A Fig). We then cloned three of these homologs and tested their interactions with LRD6-6. The result showed that at least the protein encoded by Os12g02830 could interact with LRD6-6 (S30B Fig), suggesting that Os12g02830 likely functions redundantly with OsSNF7. Thus, to elucidate the role of OsSNF7 and its homologs in immunity and cell death in rice, we may need to develop double or multiple knock-out mutants of these genes in future studies.
Another core component of the ESCRT-III complex, VPS2, is also MVBs-localized and interacts with the AAA ATPase AtSKD1 in Arabidopsis [82,83]. Three VPS2 homologs, encoded by Os03g43860, Os07g13270 and Os11g47710, respectively, were predicted in rice, based on alignment analysis of protein sequences [84]. To determine if these rice VPS2s interact with LRD6-6, we cloned full length CDSs of these genes into the vector pGADT7 to obtain pGADT7-Os03g43860, pGADT7-Os07g13270, and pGADT7-Os11g47710, respectively, and used them for Y2H tests. The result showed that the VPS2 homologous protein (we named it OsVPS2) encoded by Os03g43860 interacted with LRD6-6 (S31A Fig). BiFC assay showed that the N. benthamiana cells co-expressing OsVPS2 and LRD6-6 displayed punctate fluorescence signals with a similar pattern as LRD6-6 (S31B Fig) which further confirms the interaction between LRD6-6 and OsVPS2. These results also confirm that LRD6-6 localizes in MVBs.

Discussion
The AAA ATPase LRD6-6 inhibits immunity and cell death in rice Rice lrd mutants usually exhibit immunity-mediated cell death [48]. The lrd6-6 mutant possessed highly activated biosynthesis of antimicrobial metabolites with accumulated lignin and phytoalexins, which have been proven to fight against pathogens in rice and other plants [71,80,85,86] (Figs 9, 10, S24 and S25). Consistently, like most lrd mutants, lrd6-6 displayed accumulation of hydrogen peroxide and spontaneous cell death (Figs 1B and S4) which are usually associated with the immune response. Indeed, the immunity was enhanced in the lrd6-6 mutant as the expression of the genes associated with immunity, including the PR, chitinase, WRKY transcription factors, MPKs and oxidation-related genes, were remarkably upregulated compared with wild type Kitaake (Figs 1C and S23, S3 Table). The enhanced immunity and cell death in the lrd6-6 mutant thus led to increased resistance to the pathogens M. oryzae and Xoo (Figs 1D, 1E and S5). Further analyses identified a novel AAA ATPase LRD6-6, whose disruption resulted in the lrd phenotype in the lrd6-6 mutant (Figs 2 and S9). Thus, the ATPase LRD6-6 negatively regulates immunity and cell death in rice. A very recent study reported cloning of the Lmr gene, the same gene as Lrd6-6, which harbors a G to A base substitution resulting in a premature translation termination in the lmr mutant [51]. The lmr mutant also showed cell death and displayed enhanced disease resistance to pathogens like the lrd mutant. However, this previous study did not characterize this protein biochemically or elucidate the underlying regulatory mechanism [51].
Previous studies have isolated about 14 genes responsible for lrd phenotypes and exhibit immunity-mediated cell death. Of them, only SPL28 is associated with protein trafficking. However, the Golgi apparatus-localization suggests that SPL28 may specifically be involved in the post-Golgi trafficking process [26]. Thus none of these proteins are associated with ATPase, MVBs-mediated vesicular trafficking as is LRD6-6. Together, these suggest that the molecular regulation of immunity and cell death in these lrd mutants are very complicated and the regulatory machinery in the lrd6-6 mutant differs from those in other lrd mutants characterized previously.
The LRD6-6 protein targets MVBs, and the MVBs-mediated vesicular trafficking is associated with rice immunity and cell death Many AAA ATPases belong to the same subfamily as LRD6-6, including the mammal SKD1 [57], yeast Vps4p [58], ice plant mcSKD1 [87] and Arabidopsis AtSKD1 [38], which spread mainly on MVBs and are required for MVBs biogenesis, and its mediated vesicular trafficking [60]. In the regulation of MVBs biogenesis, the AAA ATPase of this subfamily is recruited to the MVBs membrane by ESCRT-III subunits. Then, the AAA ATPase provides the energy to disassociate the ESCRT-III complex from the membrane, and this has been characterized as the last essential step of MVBs biogenesis [60]. When the AAA ATPase is mutated or its function is rendered dominant-negative by interaction with its catalytically inactive EQ mutant, both the MVBs biogenesis and its mediated vesicular trafficking are largely blocked [60]. This can even be lethal in Arabidopsis [38,45], and suggests the conserved roles of these AAA ATPases in diverse species [36]. A recent study reported that LMR, encoded by the same gene Lrd6-6, localizes in chloroplasts for its function [51]. However, our study revealed that the LRD6-6 protein spreads on MVBs (Fig 3), which is in agreement with the MVBs-localization of the AAA ATPases from this family [38,57,58,88]. Firstly, the protein LRD6-6 fused with GFP and RFP on its C-terminus and with YFP on its N-terminus all displayed a punctate distribution and co-localized with the MVBs-localized marker protein RabF1/ARA6 but did not overlap with the chlorophyll auto-fluorescence (Figs 3 and S14). Based on our results, the subcellular localization pattern of LRD6-6 is similar to its Arabidopsis homolog AtSKD1, previously shown to be MVBs-localized [38]. Secondary, LRD6-6 co-localized and interacted with ESCRT-III components OsSNF7 and OsVPS2 (Figs 11, S30 and S31), which are respectively homologous to the MVBs-localized AtSNF7 and AtVPS2 from Arabidopsis [82,84]. Taken all together, we thus conclude that LRD6-6 mainly localizes on MVBs.
The MVBs-localization suggests that the LRD6-6 AAA ATPase is associated with MVBsmediated vesicular trafficking in rice. Indeed, although this trafficking machinery is not generally affected (S22 Fig), it is dysregulated in the lrd6-6 mutant as revealed by whole transcriptome expression analyses and the inhibited trafficking of the soluble vacuolar cargo AtCPY from ER to vacuoles (Figs 8, S20 and S21, S2 Table). Both the genes that likely encoding components of secretory and endocytic trafficking are co-regulated. For example, the AP-3β-coding gene Os01g74180 was down-regulated; the clathrin heavy chain coding gene Os12g01390 was up-regulated in both lrd6-6 and Kitaake plants expressing Lrd6-6 E315Q (S20 and S21 Figs, S2 Table). It is thus likely that both these two trafficking pathways are dysregulated in plants with loss of function of the AAA ATPase. Thus, our study demonstrates the essential roles of the AAA ATPase LRD6-6 in MVBs-mediated vesicular trafficking similar to SKD1 [57] and its homologous proteins AtSKD1 from Arabidopsis [38,45], VPS4p from yeast [58]. Our study also supports the notion that the AAA ATPases of the SKD1 subfamily are conserved in function in diverse species [60]. Importantly, our study reveals that the AAA ATPase LRD6-6 inhibits the immune response, suggesting that the LRD6-6-mediated modulation of immunity and cell death is associated with MVBs-mediated vesicular trafficking in rice, which is not reported previously for SKD1 or any other homologous proteins. Although the dysregulated MVBs-mediated vesicular trafficking may cause many biological defects, such as impeded cell proliferation, adhesion and drug resistance in humans [41], there are few reports concerning their regulation of immunity and cell death. Previous studies have implied that cell death may occur in plants with AtSKD1 knocked out or a catalytically inactive variant dominant-negative mutant AtSKD1 E232Q in Arabidopsis [38,45]. However, no direct evidence was provided from their studies because AtSKD1 knock-out or expression of AtSKD1 E232Q is lethal to plants [38,45]. Interestingly, the rice plants with disrupted LRD6-6 or dominant-negatively regulated by the catalytically inactive variant LRD6-6 E315Q still grow well, with the only visible defect of spontaneous cell death phenotype (Figs 1A and 7B). Thus, our discovery that the AAA ATPase LRD6-6 is essential for MVBs-mediated vesicular trafficking provides a valuable way to study its regulation on immunity and cell death. Of great importance, if introducing the catalytically inactive variant LRD6-6 E315Q with dominant-negative effect engineered under the pathogeninducible promoter into rice plant, we may obtain the rice with enhanced disease resistance without affecting rice yield.
MVBs-mediated vesicular trafficking has attracted much attention because of its role observed in immunity recently [31,32]. The animal pattern recognition receptor TLR4 (tolllike receptor 4) mediates perception of bacterial-derived lipopolysaccharides, and undergoes internalization upon activation with its cognate ligand through MVBs-mediated vesicular trafficking [89]. In Arabidopsis, a transmembrane leucine-rich repeat receptor kinase FLS2 that recognizes bacterial flagellin, similarly exhibits ligand-stimulated endocytosis [46,89]. The trans-Golgi network/early endosomes component KEG is reported to play a role in plant immunity by regulation of intracellular trafficking processes, and the secretion of apoplastic defense proteins [90]. VPS35B in Arabidopsis is part of the retromer complex, which functions in endosomal protein sorting and vesicular trafficking, contributing to TIR-NB-LRR and CC-NB-LRR protein-mediated autoimmunity and HR cell death [91]. However, it remains unclear how these immune proteins are transported through MVBs-mediated vesicular trafficking. In rice, trafficking is essential for the OsCEBiP/OsCERK1-OsRacGRF1-OsRac1 module to regulate immunity [92,93], while little is known about the regulation of MVBsmediated vesicular trafficking on immunity. Our study uncovered that the inhibitory regulation of AAA ATPase LRD6-6 on MVBs-mediated vesicular trafficking may be associated with plant immunity and cell death. This discovery indicates that certain immune responseassociated protein(s), including OsCEBiP in PTI, NBS-LRR proteins in ETI, and important regulators downstream, may not be sorted or transported properly due to dysregulated MVBsmediated vesicular trafficking. The disordered sorting or failure in the transport of these immune response-associated proteins may then activate the immune response without pathogen infection and results in spontaneous cell death in rice. Alternatively, it is also likely that the LRD6-6 mediated-MVBs trafficking potentially is guarded by certain NBS-LRR proteins and constitutes a downstream component of the ETI pathway. The dysfunction of this process may bypass the activation of NBS-LRR and trigger the NBS-LRR-mediated ETI response, resulting in spontaneous cell death and enhanced disease resistance similarly as reviewed previously [94]. In addition, the MVBs pathway is positively regulated by pathogen responsive MPK3/6 through phosphorylation of LIP5, an ESCRT component [46]. As MPK3/6 is part of PTI and ETI responses, these results seem to support the notion that regulation of MVBs trafficking is part of the ETI/PTI pathways. It is unclear how and where serotonin, lignin, and phytoalexins accumulate in the lrd6-6 mutant. It is possible that the blockage of transport of these antimicrobial compounds to vacuoles prevents their turnover in vacuoles and results in buildups in other compartments of the cell. Our results suggest MVBs-mediated trafficking may be essential for accurate delivery of these antimicrobial compounds.
Previous studies have reported that other AAA ATPases of different subfamilies to LRD6-6 are also associated with immunity in mammals and plants. The human AAA ATPase p97/VCP regulates antiviral immunity through binding directly to multi-ubiquitin chains and unfolding ubiquitin-fusion degradation substrates, such as the larger substrate adenovirus particle [39]. Overexpression of the mitochondrial outer membrane-localized AAA ATPase AtOM66 can constitutively induce salicylic acid-related defense response and cell death in Arabidopsis [44]. The tobacco AAA ATPase NtAAA1 inhibits innate immunity by regulating ethylene-and salicylic acid-mediated defense response through interaction with a small GTPase, NtARF [42,43]. However, our present study reveals that dysregulation of MVBs-mediated vesicular trafficking by disruption of the AAA ATPase LRD6-6 results in accumulation of antimicrobial metabolites which then leads to activation of immunity and cell death in rice (Fig 12). This differs from the molecular regulation of immunity mediated by those AAA ATPases reported previously and defines a novel regulatory machinery of immunity in plant. Thus, our discovery provides novel insights into immunity regulated by the AAA ATPase LRD6-6 likely through MVBs-mediated vesicular trafficking in rice and possibly other species.

Plant materials and growth conditions
The lrd6-6 mutant was obtained from the tissue cultured rice, Kitaake. The lesion spot appeared about 15 days after sowing. Husked seeds of Kitaake and the lrd6-6 mutant were sterilized in 30% bleach for 30 min followed by rinsing three times with sterile ddH 2 O. To investigate whether the lesion spot phenotype occurs under sterile conditions, the sterilized seeds were germinated on ½ Murashige and Skoog (MS) medium in SoLo cup and incubated in growth chamber until presence of lesion spots on leaves. For other phenotypic characterizations and map-based cloning, the plants were grown in the fields at Sichuan Agricultural University in Wenjiang, Chengdu, or Lingshui, Hainan, China.

Shading treatment
The leaves of both the lrd6-6 mutant and Kitaake plants were shaded by bandaging with silver paper before initiation of lesion spot in lrd6-6 until clear presence of lesion spots on the part of leaf without bandage.

Chlorophyll content measurement
The leaves from the lrd6-6 mutant and Kitaake plants were collected at three days before and after lesions appearance, respectively. Samples with 200 mg leaf tissue of each were soaked in 30 ml 80% acetone for 48 h in dark until the disks became colorless. Chlorophyll concentration was measured with four experimental repeats following the method as described previously [95].

Histochemical analysis
Trypan blue staining assay was performed on fresh leaves following the method as described previously [96]. In brief, samples were submerged in lactic acid-phenol-trypan blue solution (2.5 mg/ml trypan blue, 25% (w/v) lactic acid, 23% water-saturated phenol and 25% glycerol in H 2 O) and were boiled in water for 2 min, then de-stained with solution containing 30% (w/v) chloral hydrate for 3 days with multiple exchanges of the solution. After distained completely, the samples were then equilibrated with 50% glycerol for five hours followed by photo-picture taken. Detection of H 2 O 2 accumulation was carried out using DAB staining method as described previously [97]. Briefly, leaf samples were immersed in 1 mg/ml DAB containing10 mM MES (pH 6.5) for 12 h in the dark at 30°C. Then the leaf samples were transferred to solution containing 90% ethanol and 10% glycerol at 90°C until chlorophyll was completely removed. Cleared leaves were examined and photographed using an Olympus anatomical lens.

Electron microscopy
The samples were collected from the leaves of the lrd6-6 mutant and Kitaake plants before and after appearance of lesion spot in lrd6-6. The samples were then prefixed with a mixture solution of 3% glutaraldehyde. Subsequently, post-fixed in 1% osmium tetroxide, dehydrated in series acetone, infiltrated in Epox 812 for 4 hours, and embedded [98]. The sections were stained with methylene blue and ultrathin sections were cut with diamond knife, stained with uranyl acetate and lead citrate. Sections were examined under a transmission electron microscope (TEM; HITACHI, H-600IV, Japan).

RNA isolation and qRT-PCR
The mRNA samples were extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) following the procedures as described by the manufacturer. The mRNA was treated with DNase I according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA) and was subject to reverse transcription to synthesize first-strand cDNA. Oligo (dT) was used as primer and SuperScript II (Invitrogen, Carlsbad, CA, USA) was used as reverse transcription enzyme.
The qRT-PCR was conducted using a Bio-Rad CFX96 Real-Time System coupled to a C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA). The reference gene Ubiquitin 5 (Ubq5) [99] was used as control for qRT-PCR experiments. The sequences of the primers were listed in S6 Table. Determination on the disease resistance of the lrd6-6 mutant to rice blast and bacterial blight diseases Ten-days-old seedlings of Kitaake and the lrd6-6 mutant were used for inoculations with Magnaporthe oryzae (M. oryzae). The M. oryzae strains, ZB25, Zhong1 and ZE-1, which are compatible with Kitaake, were used for inoculation. Spore concentration was adjusted to 2×10 5 spores/ml with a hemacytometer before spraying [100]. The disease lesion length and the sum of lesions were acquired at day seven after inoculation.
The Xoo strains, P2, P4, P5, P6 and Xoo-4, which are compatible with Kitaake, were used for inoculation. Xoo bacterial suspensions with 0.5 of OD 600 were used to inoculate by using the scissors-dip method as described previously [101]. Disease lesion lengths were determined at day 15

Genetic analysis and map-based cloning of the locus Lrd6-6
Three F 1 s and three F 2 populations derived from the crosses of Jodan × lrd6-6, lrd6-6 × Jodan and 02428 × lrd6-6 were respectively used for the genetic analysis. The F 2 population derived from the cross of 02428 × lrd6-6 was used for mapping of the gene, lrd6-6. Bulk segregation analysis (BSA) was first used to rapidly locate the locus of lrd6-6 on a chromosome. For the BSA analysis, equal amount of leaf blades from six F 2 plants with the lesion spot phenotypes or six F 2 plants with the wild type phenotypes were collected for DNA extraction to construct the mutant and the wild type DNA pools, respectively. The physical linkage map was then constructed using additional markers nearby the locus of lrd6-6.
The SSR primers were synthesized according to the information from Gramene database (http://www.gramene.org/microsat). InDel markers were designed based on the alignment analyses on the reference japonica rice Nipponbare (http://www.rgp.dna.affrc.go.jp) and indica rice 93-11 (http://www.rise.genomics.org.cn) genome sequences at the target location. Primers were designed using Primer 5 software. The specificity of each primer in the rice genome was confirmed by BLAST and PCR analyses.
For analysis of the PCR product, amplified products by the primer pairs of the markers, I4-2, RM19274, RM19320, I8, were separated by 3.5% agarose gel electrophoresis in 1×TAE buffer, and visualized and photographed under UV light. The amplified products by RM8075 and RM587 were separated by 6% denaturing polyacrylamide gel electrophoresis and visualized by silver staining according to the method described previously [102].
For whole genomic resequencing, lrd6-6 was backcrossed with Kitaake for twice and selfcrossed to produce BC 2 F 2 progenies homozygous for the locus of lrd6-6. Equivalent total DNA of 30 BC 2 F 2 plants homologous for lrd6-6 locus were pooled and sequenced at Beijing Genomics Institute (BGI, Beijing, China). For genome sequence comparison, the Kitaake genome DNA was also sequenced.

Plasmid constructs
For RNA interference (RNAi) construct, a unique cDNA fragment of Os06g03940 from 557 bp to 1002 bp was amplified and put into the pANDA vector [103] to create RNAi construct, pANDA-Os06g03940Ri, using LR recombination enzyme (Invitrogen, Carlsbad, CA, USA).
For genetic complement construct, the genomic DNA fragment of 11.5 kb containing the native promoter (1960 bp upstream of ATG), entire coding region of LOC_Os06g03940 (Os06g03940) and a fragment with 2295 bp downstream of TAG, was cloned into the binary vector pCactN-XG following the steps: I, Four DNA fragments covering the 11.5 kb genome sequence from Nipponbare genome were amplified with KOD DNA polymerase (Toyobo, Osaka, Japan) using the primers, lrd6-6cp-P1, lrd6-6cp-P2, lrd6-6cp-P3, lrd6-6cp-P4, listed in S5 Table. These fragments were then cloned into the vector pEASY Blunt Simple (TransGen Biotech, Beijing, China) to create pSimple-CP1, CP2, CP3, CP4 respectively followed by sequencing verification (Sangon Biotech, Shanghai, China). II, the DNA fragment of CP4 was released from pSimple-CP4 under digestion with the enzymes, NdeI and SalI, and put into pSimple-CP3 pre-digested by the same enzymes to generate pSimple-CP3-CP4. III, the DNA fragment of CP1 was cut from pSimle-CP1 with BamHI and XhoI, and put into pSimple-CP2 predigested to create pSimple-CP1-CP2. IV, the DNA fragment of CP1-CP2 was digested from pSimle-CP1-CP2 with the enzymes, BamHI and SmaI, and put into TSK108 vector predigested to get TSK108-CP1-CP2. V, the DNA fragment of CP3-CP4 was released from pSimle-CP3-CP4 by cutting with SmaI and SalI and cloned into TSK108-CP1-CP2 to generate an intermediate vector TSK108-CP1-CP2-CP3-CP4 (TSK108-Os06g03940-11.5kb). VI, the DNA fragment of Os06g03940-11.5kb was then cut from TSK108-Os06g03940-11.5kb with BamHI and SalI and cloned into the predigested pCactN-XG to get the destination construct pCacTN-XG-Os06g03940-11.5kb. The construct pCacTN-XG-Os06g03940-11.5kb was verified by sequencing before used for transformation (Sangon Biotech, Shanghai, China). For other constructs, the intended segments were cloned into the vector pEASY Blunt Simple and then subcloned into the destination vectors. All the constructs information and the primers used were listed in S5 Table.

Rice genetic transformation
The Agrobacterium-mediated transformation was used for rice genetic transformation according to the method described previously [104]. The transgenic plants obtained were selected with Hygromycin or G418 during regeneration. The positive transgenic plants were then verified by PCR-based genotyping using the primer pairs, specific for hygromycin gene or Neomycin phosphotransferase II gene (NPT II) selected with G418, respectively.

Subcellular localization
For subcellular localization in Nicotiana benthamiana (N. benthamiana) and onion epidermal cells, the plasmid DNA of p35S:Lrd6-6-GFP was introduced into N. benthamiana leaf by agroinfiltration [105] and onion epidermal cells using a bombardment-mediated gene transformation [106], respectively. The N. benthamiana leaf and onion epidermal cells were transformed with p35S:GFP as controls. Fluorescence was examined under a confocal microscopy (NiKon A1 i90, LSCM, Japan) 36 h post transformation of N. benthamiana and 16 h post transformation of onion epidermal cells, respectively.
For subcellular localization in rice protoplasts, the plasmid, pBI221-AtCPY, was transformed into protoplasts prepared from Kitaake and lrd6-6 mutant seedlings following the method as described previously [107]. For a control, the protoplasts were transformed with pBI221. Fluorescence was examined under a confocal microscopy (NiKon A1 i90, LSCM, Japan) 16 h after transformation.

Wortmannin treatment
The N. benthamiana leaf expressing the RabF1/ARA6-GFP or RabF1/ARA6-RFP fusion protein was immersed into solution containing 33 μM wortmannin (Selleckchem) for 40 min and then examined under a confocal microscopy similarly as described above.
For treatment of the rice protoplasts expression AtCPY-GFP, wortmannin (33 μM) was added into the incubation buffer 4 hours after transformation and then incubated at 28°C until examination.

Protein extraction and immuno-blot analysis
The N.benthamiana leaves expressing the interested proteins were respectively harvested 48 hours post transformation. Protein extraction and immunoblotting analysis with corresponding antibodies as indicated were performed according to the method described previously [105].
For determination of the protein express in yeast, the total protein extract was prepared and analyzed with anti-Myc and anti-HA antibodies, separately, following the Yeast Protocols Handbook from Clonetech (Otsu, Shiga, Japan).

Site directed mutagenesis of Lrd6-6
To obtain the mutants, K261A, E315Q and R372E of LRD6-6, the full-length coding sequence of Lrd6-6 was cloned into the vector pEASY Blunt Simple. Mutagenesis was then performed with the specific primers (Listed in S5 Table) using QuikChange site-directed mutagenesis kit following the manual (Stratagene, La Jolla, California, USA) and the intermediate cloning and final constructs harboring the desired mutation sites were verified by sequencing.

Protein expression, purification and ATPase assay
The truncated LRD6-6 (AAs: 125-487, covering the ATPase domain) and its mutation versions were cloned into the pET28a vector (Novagen), and expressed in the E.coli strain BL21. Bacteria contain the plasmids were grown in Luria-Bertani (LB) medium containing 100 μg/ml and kanamycin at 37°C to OD600 = 0.6, induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to final concentration of 1 mM and incubated at 28°C for 6 hours respectively. Cells were pelleted by centrifugation, re-suspended in lysis buffer ( ATPase activity of LRD6-6(125-487), LRD6-6(125-487) K261A , LRD6-6(125-487) E315Q and LRD6-6(125-487) R372E were measured by the malachite green-based colorimetric method using the ATPase/GTPase activity assay kit (Sigma-Aldrich, St. Louis, MO, USA). The elution buffer was used as negative control. One unit is termed as the amount of enzyme that catalyzes the production of 1 μM of free phosphate per minute under the assay conditions.

Yeast two-hybrid assay and library screen
Yeast two-hybrid assay was performed using the GAL4-based Matchmaker Gold Yeast Two-Hybrid System (Cat. No. 630489, Clontech, Otsu, Shiga, Japan), in which the bait protein is expressed as a fusion to the Gal4 DNA-binding domain in pGBKT7 vector and the prey protein is expressed as fusion to the Gal4 activation domain in pGADT7 vector [108,109]. Full-length amino acids of the proteins tested were cloned in frame with the Gal4 DNA-binding or activation domain using genes specific primers to obtain the constructs (Listed in S5 Table). After sequence verified, the constructs were co-transformed by pair into the yeast stain Y2HGold via polyethylene glycol (PEG)/LiAc-based method provided by the Yeastmaker Yeast Transformation System 2 (Cat. No. 630439, Clontech). The transformed Y2HGold cells were plated on DDO media (double dropout medium: SD/-Leu/-Trp) and the cell clones were diluted for 3 gradients on the QDO/A media (quadruple dropout medium: SD/-Ade/-His/-Leu/-Trp supplemented with Aureobasidin A) for interaction test.
After confirmation that no autoactivation and toxicity exist in the Y2HGold cells co-transformed with pGBKT7-LRD6-6 and the null pGADT7 vector, yeast two hybrid screening was performed by co-transformation with the pGBKT7-LRD6-6 plasmid DNAs (10 μg) and cDNA library DNA (fused into the pGADT7 vector) (10 μg) were co-transformed into the Y2HGold cells following the procedure in the manual of Yeastmaker Yeast Transformation System 2 (Cat. No. 630439, Clontech). Plasmids of the positive clones grew on the selective QDO/A media were rescued and were respectively co-transformed with pGBKT7-LRD6-6 or the empty bait vector pGBKT7 into Y2HGold cells to validate the interactions. After verification of the interactions, the plasmid DNAs of clones were subject to sequencing.

Bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana
The full-length coding sequences of Lrd6-6, lrd6-6, Lrd6-6 K261A , Lrd6-6 E315Q , Lrd6-6 R372E and OsSnf7 without stop codon were amplified using specific primers (Listed in S5 Table) and cloned into the psPYNE and/ or psPYCE vectors through XbaI and BamHI to generate their respective YFP N and/ or YFP C fusion protein expressing constructs [110]. Equivolume suspensions of different Agrobacterium strains carrying different constructs were mixed prior to infiltration and co-infiltrated into N.benthamiana leaves following the method described previously [105]. Fluorescence was examined under a confocal microscopy (NiKon A1 i90, LSCM, Japan) 36-48 hours post transformation.
Genome-wide transcript analysis on the lrd6-6 mutant and Kitaake When lesion spot initiated on leaf of the lrd6-6 mutant, the leaf tissues from lrd6-6 and the equivalent part of Kitaake as indicated (S18 Fig) were sampled for RNA extraction. RNA-seq was performed by CapitalBio Corporation at Beijing, China. Cuffcompare [111] was used to compare the assembled transfrags of each library to the reference annotation, and build up a non-redundant transcripts data set among the libraries. Then Cuffdiff [111] was used to identify DEGs. Transcripts data set were first compared with Kyoto Encyclopedia of Genes and Genomes database (KEGG, release 5.8) [112] using BLASTX [113] at E values < = 1e-10. A Perl script was used to retrieve KO information from blast result and then established pathway associations between transcripts and database. InterPro domains [114] were annotated by InterProScan (release 4.8) [115] and functional assignments were mapped by using GO (Gene Ontology) analysis [116]. Expression verification of the selected DEGs was conducted by using q-RT-PCR analysis. The primer pairs used were listed in S5 Table. Determination of the lignin, total SA and phytoalexins content in rice The lignin content and total SA content were determined according to the acid detergent lignin method and the ultrahigh performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS) method as described previously [117,118].
For determination of phytoalexins, tissue samples (ca. 10 mg fresh weight for each) were harvested from the lrd6-6 mutant and Kitaake. The samples were soaked in 1 ml extraction solvent (MeOH/H 2 O, 80:20 [v/v]) and incubated at room temperature overnight. Then, 5 μL of the extract was subjected to phytoalexin measurement by LC-ESI-MS/MS. An Agilent 1200 separation module (Agilent Technologies, Palo Alto, CA, USA) equipped with a CAPCELL CORE C 18 column (50 mm long, 2.1 mm in diameter; Shiseido, Tokyo, Japan) was used for HPLC analysis. The mobile phase consisted of 0.05% AcOH in H 2 O (solvent A), and 0.05% AcOH in MeCN (solvent B). Elution was conducted using a linear gradient from 40% to 60% solvent B over 10 min at a flow rate of 0.2 mL/min, and the eluate monitored by Agilent 6460 Triple Quadrupole mass spectrometer (Agilent). The ionization mode was electrospray. All phytoalexins were analyzed in positive ion mode. Electrospray conditions were as follows: capillary voltage, 3500 V; drying gas flow, 5 L/min nitrogen; drying gas temperature, 300°C; nebulizer pressure, 45 psi; sheath gas temperature, 350°C; and sheath gas flow, 11 L/min.