Characterization and Proteomic Analysis of Novel Rice Lesion Mimic Mutant with Enhanced Disease Resistance

Yang Yong, Lin Qiujun, Chen Xinyu, Liang Weifang, Fu Yuwen, Xu Zhengjin, Wu Yuanhua,Wang Xuming, Zhou Jie, Yu Chulang, Yan Chengqi, Mei Qiong, Chen Jianping, ,

Research Paper

Characterization and Proteomic Analysis of Novel Rice Lesion Mimic Mutant with Enhanced Disease Resistance

Yang Yong1, #, Lin Qiujun2, #, Chen Xinyu3, Liang Weifang4, Fu Yuwen2, Xu Zhengjin2, Wu Yuanhua2,Wang Xuming1, Zhou Jie1, Yu Chulang5, Yan Chengqi6, Mei Qiong2, Chen Jianping1, 2, 5

(; ; ; ; I; These authors contributed equally to this work)

Lesion mimic mutants (LMMs) are plants that spontaneously form lesions without pathogen infection or external stimulus and exhibit resistance to pathogens. Here, a rice LMM was created by ethyl methane sulfonate mutagenesis, named as(hydrogen peroxide induced lesion). Diaminobenzidine and trypan blue staining showed that large amounts of H2O2were produced and cell death was occurredat and around the parts of lesion mimic in the rice leaves. The phenotype ofis controlled by a single recessive gene, localized at a 2 Mb interval on chromosome 2. The data suggested thatis a novel LMM with enhanced bacterial and fungal disease resistance, and multiple pathogenesis-related proteins(PRs)were up-regulated. The proteomes of leaves at three positions (different degrees of lesion mimic severity) were characterized incompared with its wild type plant. Differentially expressed proteins were detected bytwo dimensional difference gel electrophoresis and 274 proteins were identified by MALDI TOF/TOFTM. These proteins were related to metabolic process, cellular process and response to stimulus,with mostly down-regulated inleaves. Many of these proteins were related to the Calvin cycle, photosynthetic electron transport chain, glycolysis/gluconeogenesis and phosphonates pathways. Some resistance-related proteins including 14-3-3 proteins, OsPR10 and antioxidases such as peroxidase, superoxide dismutaseand ascorbate peroxidase were up-regulated in leaves with lesion mimic. These results provide the foundation for cloning of the target gene and shed light on the mechanism involved in autoimmunity of rice.

lesion mimic mutant; H2O2;disease resistance;pathogenesis-related protein; resistance- related pathway

Programmed cell death (PCD) is a controllable gene regulation process that may occur spontaneously or in response to an external stimulus. PCD is connected with development as well as biological and abiotic stress. The hypersensitive response (HR) is a key characteristic of PCD in plants (Coll et al, 2011). It can induce cell death at and around an infection site to limit further pathogen infection (Dangl et al, 1996). Reactive oxygen species (ROS) and plant hormones such as salicylic acid, jasmonic acid and ethylene play an important role in the occurrence and regulation of HR. Under normal conditions, plants maintain ROS homeostasis, but the balance is disturbed by biological and abiotic stresses, which can lead to ROS accumulation. Most incompatible interactions between pathogens and plants can induce an oxidative burst,resulting in PCD at the infection site and in surrounding cells. In this process, ROS reacts with most cell compounds,resulting protein degradation, mutagenesis of DNA, oxidation of purines, and the crosslinking of proteins and DNA (Beckman and Ames, 1997). Thus, high level of ROS can lead to typical symptoms of cell death. ROS is not only directly toxic to cells but also can induce a signal cascade reaction leading to PCD (Suzuki et al, 2011; Waszczak et al, 2018).

Plant lesion mimic mutants (LMMs) can spontaneously form cell death spots in the absence of pathogens or any external environment stimulus (Bruggeman et al, 2015). LMMs are often resistant to various pathogens and therefore excellent for studying PCD and immune responses (Zhu et al, 2020). LMMs are of two types, known as constructive PCD (initiation mutant) and uncontrolled PCD (propagation mutant) (Lorrain et al, 2003). Initiation mutants form controllable cell death spots on a small scale whereas the PCD in propagation mutants is uncontrollable and ultimately leads to the death of the whole plant. At least 55 LMMs have been characterized and no less than 21 target LMM genes have been cloned so far. These LMM genes encode diverse proteins with many different biological functions, showing that various pathways might be involved in the occurrences of lesion mimic. For example, CC-NBS-LRR-type protein participates in salicylic acid or NPR1-mediated resistance signaling in rice(Tang et al, 2011). Zinc finger proteins positively regulate callus differentiation and negatively regulate cell death in tobacco cells(Wang et al, 2005). E3 ubiquitin ligase genes such as(Zeng et al, 2004) and(Liu et al, 2017) are involved in PCD.confers not only resistance to rice blast (caused by) but also resistance to rice bacterial blight [caused bypv.()], and the resistance is related to the expression of disease resistance genes and the size of disease spots on leaves (Yin et al, 2000). SPL11 has a U-box domain and an ARM (armadillo) repeating domain, which play roles in ubiquitination and protein interactions in yeast and mammalian systems, respectively.is induced by the incompatibility of the rice-interaction.ubiquitination analysis showed that SPL11 has E3 ubiquitin ligase activity that depends on its complete U-box domain, suggesting that the ubiquitination system plays a role in controlling plant cell death and disease resistance (Zeng et al, 2004). In another E3 ubiquitin ligase class lesion mutant, ROS production and the expression of related genes induced byand chitin significantly enhance resistances to rice blast and bacterial leaf blight. OsCUL3a negatively regulates cell death by degrading OsNPR1 (Liu et al, 2017). Some LMM genes encode membrane related proteins which play an important role in plant disease resistance, such as receptor-like protein kinases that have an extracellular leucine-rich repeat (eLRR) domain, a transmembrane domain and a cytoplasmic kinase domain. OsLRR1 has a simple eLRR domain, and interacts with the rice hypersensitive-induced response protein 1 (HIR1), while the two homologous proteins, LRR1 and HIR1, are highly conserved in different plants (Zhou et al, 2010). Afterwas transferred into, it spontaneously induced lesion mimic, thus enhancing the immunity to bacteria. In addition, genes encode eukaryotic extension factors (eEF) or release factors (eRF) such as/and(eEF1A genes), and/(eRF1 genes) provide resistance to several pathogens includingand(Wang et al, 2017; Zhao et al, 2017; Qin et al, 2018; Zhang et al, 2018)LMM5.1/SPL33 and LMM5.4 have 97.4% amino acid sequence identity and are functionally redundant.

We identified a LMM calledcreated by the ethyl methane sulfonate (EMS)from rice cultivar Zhejing 22 (ZJ22).has typical characteristics of autoimmunity including PCD and ROS accumulation. It was proved to be highly resistant toand. Five PRs (,,,and) were significantly up-regulated in. We mapped the target gene to a 2Mb interval on chromosome 2. Proteomics tests on leaves with different degrees of lesion mimic ofand wild type plant revealed various proteins closely linked to autoimmunity in rice.

Results

Phenotype of hpil mutant

Young seedlings ofdid not have any lesions just like the wild type plants, and subsequently the leaves gradually developed reddish-brown lesions from two weeks after seed germination. Over time, the number and size of spots increased until the whole leaf was covered (Fig. 1-A and-B),but the shaded region of leaves showed no lesion mimic (Fig. 1-B).

Cell death and ROS accumulation in hpil

Trypan blue (TB) stains were used to detect death cells. The results showed that various degrees of cell death occurrence in lesion mimic leaves ofin contrast to the wild type plants(Fig. 1-C),while the shaded regions ofleaves showed no lesions which was similar to the wild type plants.

ROS such as H2O2possesses durable function and is often as resistance signal molecules and antibiotic substances that are usually accumulated in plant cells with PCD. Diaminobenzidine (DAB) reacts with H2O2to form a reddish-brown precipitate, which indicates the site of H2O2production. In our study, compared with the wild type plants,except for the shaded region of leaves had abundant ROS accumulation in and around the lesion mimic (Fig. 1-D).

Resistance analysis

Many LMMs are resistant to multiple fungal and bacterial pathogens (Fekih et al, 2015; Wang et al, 2015). In our study, we inoculatedand wild type plants with 10racesand 2races. The results showed thatpresented highly resistant to all these races compared to the susceptible wild type plants (Fig. 2). And five PRs tested by qRT-PCR were significantly up-regulated incompared to the wild type plants (Fig. 1-E). This demonstrated thatshows typical autoimmune characteristics.

Genetic analysis and gene mapping

To determine the inheritance pattern of the lesion mimic phenotype of, we back-crossedwith the wild type plant ZJ22. All the F1plants showed no any lesions on their leaves just like the wild type plants, and the lesion mimic phenotype isolation occurred in F2plants.Of 139 F2plants, 33 showed lesion mimic phenotype and 106 showed wild type phenotype. The F2population showed the segregation of the wild type and lesion mimic phenotypes in a ratio of about 3:1 (χ2= 0.06 < χ20.05= 3.84). This indicated that the lesion mimic phenotype ofis controlled by a single recessive gene.

To locate the mutation, we performed genome resequencing to F2back-cross group ofand ZJ22. Nipponbare rice (IRGSP-1.0) was used as the reference genome for data analysis. The consensus sequences were used to genotype the single nucleotide polymorphisms (SNPs) obtained from the F2population. We obtained 166 168 markers in the offspring pool. Taking the genotype of ZJ22 as a reference, analysis of genotype frequency index was conducted for mutant population and wild type population, respectively. To obtain the frequency index of mutant-type reads at all sites, mutant-type reads depth at each marker site was divided by the total reads depth at that site. Reads with index less than 0.3 in offspring were filtered out. The average value of mutation frequency in all ‘slide windows’ (five continuous SNPs) of each chromosome was calculated. Finally, we located the target gene within a 2 Mb interval on chromosome 2 (Fig. S1) with the F2back-cross population ofand ZJ22 using the strategy by connection of genome resequencing and map-based cloning mentioned above. Isolation and identification of the targetfor spotted-leaf phenotype is currently underway.

Fig. 1. Characterization ofand relative expression of pathogenesis-related proteins (PRs)

A, Phenotypes of Zhejing 22 (ZJ22) and.

B, Leaves of ZJ22 and. Shaded region is indicated by red box.

C, Trypan blue staining of leaves of ZJ22 and. Shaded region is indicated by red box.

D, Diaminobenzidine staining of leaves of ZJ22 and. Shaded region is indicated by red box.

E, Relative expression of PRs in ZJ22 and. Data are Mean ± SE (= 3). **,< 0.01.

Fig. 2. High resistance ofto different races ofand.

A, Leaves of Zhejing 22 (WT) andinoculated with 10 different races of.

B, Lesion lengths caused byinfection. Data are Mean ± SD (= 6); **,< 0.01.

C, Leaves of WT andinoculated with two different races of.

D, Disease severity of leaves inoculated with. Data are Mean ± SD (= 10); **,< 0.01.

Proteomic analysis

Because the resistance tohas highly correlation with the degree of lesion mimic as mentioned earlier, we compared the protein profiles ofleaves on three different positions, namely leaf A (no lesion mimic), leaf B (a spot of lesion mimic) and leaf C (more severe lesion mimic), with the leaves from the corresponding positions on wild type plants as controls. Two dimensional difference gel electrophoresis (2D-DIGE) analysis was made to isolate the differentially expressed proteins (DEPs), of which 1.5-fold change ones were picked out. DEPs(repeat proteins are excluded)identified by tandem mass spectrometric (MS/MS) are shown in Table S1 and all DEPs are shown in Table S2. Accordingly, 274 of 294 protein spots were successfully identified by MS based on the Uniprotdatabase. Among them, 68 of these proteins were up-regulated and 206 were down-regulated incompared to the wildtype plants. There were more down-regulated protein spots than up-regulated ones identified in each leaf (Fig. S2-A). DEPs were the fewest (27) in leaf A and the most (209) in leaf B. Leaf C had 173 DEPs. This probably due to various proteins degraded in leaf C. As shown in Fig. S2-B, only seven DEPs were jointly identified in leaf A, leaf B and leaf C. However, leaf B and leaf C shared 111 same DEPs which are probably related to lesion mimic. DEPs only identified in leaves A, B or C were probably related to lesion mimic at different stages.

Gene Ontology (GO)Classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) of DEPs

The 274 DEPs were grouped according to their biological processes using GO annotation. They are metabolic process (65), cellular process (53), response to stimulus (40), single-organism process (12), localization (10), cellular component organization or biogenesis (8), developmental process (8), multicellular organismal process (8), reproduction (7), reproductive process (2), biological regulation (1), multi-organism process (1), regulation of biological process (1) and signaling (1) (Fig. S3). Only up-regulated proteins in biological regulation, regulation of biological process and signal related proteins, and down-regulated proteins in multi-organism process were detected. In the cellular component category, only DEPs of membrane-enclosed lumen were down-regulated, and in the molecular function category, only proteins of molecular function regulator, nucleic acid binding transcription factor activity and structural molecule activity were down-regulated.

According to KEGG analysis, the largest category of DEPs was involved in metabolic pathways (40), followed by carbon metabolism (23) and carbon fixation in photosynthetic organisms (17). Most of these DEPs were down-regulated, showing that these protein-related metabolic pathways were suppressed. Other DEP-enriched pathways wereglyoxylate and dicarboxylate metabolism (12), photosynthesis (12), biosynthesis of amino acids (7), oxidative phosphorylation (6) and glutathione metabolism (4) (Fig. S4).

Different levels of mRNA and protein expression

We performed qRT-PCR analysis to investigate the changes of DEPs at the mRNA level (Fig. 3). Total mRNA was extracted respectively from leaf A, leaf B and leaf C of ZJ22 and. The overview of different levels of mRNA and protein expression is shown in Fig. 3. The mRNA expression levels of three 14-3-3 like proteins were lowercompared with changes in their protein levels. The mRNA of four DEPs(AAA type ATPase, OsAPX2, OsPR10 and SOD), with the relationship of the resistance, were higher compared with changes in their protein levels.

Discussion

Autoimmunity is activated in hpil

In this study, we isolated a novel lesion mimic mutantwith reddish brown lesions. The lesion mimic trait derives from a single-gene mutation on chromosome 2.displayed horizontal resistance to 10races and 2races, and 4 PR genes were up-regulated in our study.Plants have evolved animal-like innate immunity during their long battles with pathogens (Jones and Dangl, 2006) and two types of response have been identified. PAMPs-triggered immunity (PTI), a basic plant defense response, is induced by the pathogen surface associated molecular pattern (PAMP), such as specific polysaccharides, and flagellin.Effector-triggered immunity (ETI) is regarded as the second level of the immune system. In this specific process, disease-resistance protein in plants recognizes effector protein that is produced by pathogen. ETI often induces HR, which is characterized by the generation of PCD (Cheng et al, 2011; Cheng and Li, 2012; Rodriguez et al, 2015). However, PTI and ETI are not completely independent and have overlapping parts (Navarro et al, 2004; Tsuda and Katagiri, 2010). Studying oncan help reveal the mechanism of plant immune system.

Calvin cycle related proteins

In the Calvin cycle, ATP and NADPH convert carbon dioxide and water into sugars, and this process is the dark part of photosynthesis. In this study, a large number of DEPs involved in the Calvin cycle were identified (Fig. S5 andTable S2). Most of these proteins were down-regulated incompared to the wild type plants, indicating that a serious suppression of Calvin cycle was happened in. Some of these proteins, such as ribose-5-phophate isomerase and transketolase, are involved in the fixation of CO2in photosynthesis, and their reduction inhibits ribulose-1,5-diphosphate regeneration and photosynthetic reactions, resulting in the reduction of chlorophyll (Henkes et al, 2001). Compared with wild type plants, the expression level of transketolase was only down-regulated in leaf C of, indicating that these proteins were significantly down-regulated at the later stage of lesion mimic production. ATP synthase catalyzes the synthesizing of ATP from ADP in the presence of a transmembrane proton gradient. Large amount of ROS accumulation and ATP synthase were not detected in lesion mimic mutant, which may due to the severely degradation of thylakoid membrane (Kang et al, 2007). This is similar to our study, suggesting the similar signal pathway may be activated inand.

Fig. 3. Quantitative real-time PCR analysis of relative mRNA levels of differentially expressed proteins (DEPs) in leaves of WT and.

AAA type ATPase, ATPase associated with various cellular activities; SOD, Superoxide dismutase.

WT, Zhejing 22;Leaf A, The first leaf from the top with no lesion mimic; Leaf B, The second leaf from the top with mild lesion mimic; Leaf C, The third leaf from the top with severe lesion mimic.

Data are Mean ± SD (= 3).

Some proteins related to the Calvin cycle like sedoheptulose-1,7-bisphosphatase, phospho-ribulokinase,fructose-bisphosphate aldolase, triosephosphate isomerase and phospho-glycerate kinase (PGK) were down-regulated in leaf B and leaf C ofbut not in leaf A (Table S2), speculating that the Calvin cycle was suppressed in leaf B and leaf C. Ribulose phosphate 3-epimererase that plays a role in both the Calvin cycle and the phosphopentose pathway was down-regulated only in leaf B, showing that these pathways were inhibited in the mid-phase of lesion mimic.

In some plants, the overall Calvin cycle rate was decreased following pathogen infection (Akhkha and Clarke, 2003), while in other plants, only the activities of some Calvin cycle proteins, such as Rubisco and PGK, were decreased (Gordon and Duniway, 1982). PGK plays a role in the reduction phase of the Calvin cycle, thereby assisting in the biosynthesis of carbohydrates. In this study, PGK was down-regulated to one third in leaf B and leaf C of(Table S2), indicating that the appearance of lesion mimic may inhibit the biosynthesis of carbohydrates, perhaps becauseputs more energy into disease resistance. Fang et al (2012) have shown that some proteins involved in the Calvin cycle are up-regulated at 24 or 48 h after pathogen infection, which may be the response to infection, while the contents of almost entire Calvin cycle proteins are decreased at 72 h after pathogen infection, perhaps due to the PCD that prevents further infection. This result is similar to that in our study. With the degree of cell death increasing, the photosynthetic capacity of plants was gradually declined.

Photosynthetic electron transport chain related proteins

The down-regulation of several photosynthetic electron transport related proteins was detected in this study (Table S2). PsbO, PsbP and PsaE were down-regulated in leaf B and leaf C of(Table S2). Fd-NADP reductase,which is a carrier of the photosynthetic electron transport chain, was down-regulated in leaves at the three different positions of, andwas more remarkable in leaf B and leaf C. Cytochrome b6f complex, also a carrier of the photosynthetic electron transport chain, was also down-regulated in leaf B and leaf C of.

ATP synthase is composed of nine different subunits and enables ADP and Pi to synthesize ATP by using the transmembrane proton (H+) gradient formed by the energy released from the photosynthetic electron transport chain. The enzyme also catalyzes the reverse reaction, the hydrolysis of ATP and the transport of H+into the membrane of the thylakoid. The majority of the α, γ and ε subunits of ATP synthase identified in our results were down-regulated, and were usually much more severely in leaf B and leaf C than in leaf A, indicating a progressive decrease in the synthesis of ATP by photosynthesis. In addition, chlorophyll contents in leaf B and leaf C were significantly decreased in(Fig. S6). All of the effects mentioned above might lead to the decline of net photosynthetic rate (Fig. S6).

Glycolysis/gluconeogenesis and phosphopentose pathway related proteins

Glycolysis/gluconeogenesis is a central metabolic pathway. In this study, a variety of proteins associated with this pathway were considerably changed in leaves ofcompared to the wild type plants (Fig. S5 and Table S2). Fructose-1,6-bisphosphatase, fructose-biphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase and PGK were unchanged in leaf A, but were down-regulated in leaf B and leaf C (Table S2), indicating that glycolysis and gluconeogenesis were inhibited in the leaves with thelesion mimic of.

Transketolase plays an important role in the regulation of the pentose phosphate pathway, and its expression was decreased in leaf A, leaf B and leaf C of(Table S2). Ribulose-phosphate 3-epierase was down-regulated in leaf B, but showed no significant change in leaf A and leaf C. Ribose-5-phophate isomerase 3 was down-regulated in leaf C. These results indicated that the pentose phosphate pathway was inhibited in.

Disease-resistance proteins

The 14-3-3 protein is an evolutionary-conserved protein that is unique to eukaryotes. It is encoded by multiple gene families. Many studies suggest that the 14-3-3 protein regulates plant responses to pathogens. It is worth noting that some 14-3-3 proteins are target proteins for effectors of pathogenic bacteria, indicating their function in plant disease resistance (Lozano-Duran and Robatzek, 2015). In rice, some 14-3-3 proteins are induced by interactions with(GF14b, GF14c, Gf14e and GF14f) or(GF14b, GF14c and Gf14e) (Chen et al, 2006). GF14b is also induced and negatively regulated byETI and resistance of rice (Manosalva et al, 2011). Three 14-3-3 proteins identified in this study were up-regulated only in leaf B of(Fig. 3, TablesS1 andS2). This implied that these proteins may play a role in resistance and cell death. While another two 14-3-3 proteins were down-regulated at gene expression level, which is not consistent with the result of the proteomic experiment (Fig. 3 and Table S1). It could be due tothe reason that the expression of these two 14-3-3 genes were inhibited more severely or the transcribed mRNAswere degraded faster, while the 14-3-3 proteins were degraded more slowly inthan in the wild type plant. This may be related to resistance inand the mechanism needs further study.

PRs are divided into 17 classes based on amino acid sequence similarity, serological relationships and biological activity (van Loon and van Strien, 1999). The PR10 family proteins which are usually located in cells are involved in resistance to biological stress. The PR10 family proteins are involved in a variety of antibacterial activities and are usually located in cells. PR10 family proteins are involved in resistance to biological stress, such as fungi (Xie et al, 2010), viruses (Park et al, 2004), bacteria (Flores et al, 2002; Xie et al, 2010) and nematodes (da S Andrade et al, 2010). At present, four different PR10 family genes have been identified in rice:,(Mcgeeet al, 2001), jasmonic acid induced() (Jwa et al, 2001) and(root specific) (Hashimoto et al, 2004). Overexpression ofhas enhanced rice resistances toand the stress of drought and salt (Wu et al, 2016). Other studies have found thatwas induced byinoculation, and the transgenic rice overexpressingsignificantly increase their resistance toandpv.(Huang et al, 2016). JIOsPR10 identified in our study was up-regulated in leaf A and leaf B of, with expression multiples of 2.3 and 9.2 (Table S2), respectively, which may be due to the activation ofresistance in. This is consistent with qRT-PCR results (Fig. 3).

AAA protein family members usually function as chaperones, assisting protein complex assembly, operation and dissolution. They also work in stress resistance. For example, transgenic tobacco expressing SKD1 (AAA type protein of corn) is more tolerant to salt and drought stress and the accumulation of ROS in transgenic tobacco is less than that in the wild type plants(Xia et al, 2013). However, the relationship between the AAA protein family and plant disease resistance has less been studied. Fekih et al (2015) identified a lesion mimic mutantwith a mutated AAA-type ATPase. The mutant is resistant to rice blast and bacterial blight.is located in the chloroplast that is an important production site of ROS. These results indicate the mutation ofmay play a role in ROS production and cell death. Ribulose-1,5-bisphosphatecarboxylase/oxygenase activase (RCA),also an AAA protein family member,is involved in resistance ofto rice bacterial blight (Mei et al, 2019). This impliesthat RCA not only plays a role in plant resistance to abiotic stress (Crafts et al, 1997; Sharkey et al, 2001), but also works in plant resistance to biological stress. In this study, a large amount of hydrogen peroxide was produced in the leaves with lesion mimicof(Fig. 1-D). Meanwhile, most of AAA-type ATPase proteins were down-regulated in, indicating that the level of these proteins was negatively correlated with the degree of lesion mimic. This is similar to the results of the study with(Fekih et al, 2005). Additionally, the downward trend of mRNA level of the protein was high correlative with that of DEPs in leaf B and leaf C (Fig. 3). These results suggest that the AAA-type ATPase identified in our study may be involved in the ROS (produced in the chloroplast)-dependent cell death pathway.

ROS is not only an antibiotic substance but also a signal transduction molecule (Corpas et al, 2020). Superoxide dismutase (SOD) and ascorbate peroxidase (APX) reduces oxidative damage of plants by reducing ROS (Hiraga et al, 2000). SOD identified in this study was up-regulated to approximate 1.6 times in both leaf B and leaf Cofthan in those of the wild type plants. In addition, two protein spots identified as APX were up-regulated by 1.9 times in leaf B and 1.7 times in leaf C, respectively (Table S2). The mRNA levels of APX and SOD were high correlative with their protein levels (Fig. 3). The activities of SOD and APX were both increased in leaf B and leaf C of(Fig. S6). This is basically consistent to the transcriptional and translational levels. SOD transfers electrons from O2·?to H2O to generate H2O2, so the increase in the activity and the expression levels (mRNA and protein)may cause higher H2O2accumulation inthan in wild type rice. However, the up-regulation expression of APX inwas not accordant with the higher H2O2level in the mutant rice, since the role of APX is mainly to scavenge H2O2through catalyzing H2O2to produce H2O. This may be because that the amount of H2O2produced is larger than it is removed in. In addition, the damage of other H2O2scavenging pathways inmay cause the higher H2O2accumulation inthan in the wild type, which is worthy of further study. Keeping ROS homeostasis is crucial to growth and development of plant. In this study, constantly produced ROS played an important role in resistance of. At the same time, a mount of ROS was scavenged by various anti-oxidases, which preventedfrom uncontrolled cell death (the death of whole plant).

Disease resistance of hpil is light-dependent

In this study, we characterized a lesion mimic mutantwith horizontal resistant to multiple races ofand. Necrotic spots were not generated and H2O2was hardly accumulated in the shaded parts ofleaves (Fig. 1-B to -D). This demonstrated that the resistance ofis light-dependent. Spot formation in many lesion mimic mutants is related to lighting (Huang et al, 2010).encodes a zinc-finger protein and its expression is induced by light (Wang et al, 2005). Anti-sensetransgenic plants generate brown necrotic spots under short day length and exhibit increased resistance to. This is different from light-dependent resistance of.is a gain-of-function lesion mimic mutant with enhancedresistance and its lesion initiation and development is light inducible (Chen et al, 2019). Highly increased level of jasmonic acid is detected in, and this is consistent with the up-regulation of,,,and, which play an important role in jasmonic acid signaling pathway (Shen et al, 2011). In this study, the level ofwas also significantly increased in(Fig. 3)We assumed thatresistance may be initiated by the activated jasmonic acid signaling pathway in. Many photosynthesis- related proteins were down-regulated in(Table S2). Chlorophyll was also significantly degraded due to the cell death in(Fig. S6). Both results may lead to the decrease in stomatal conductance and photosynthetic rate(Fig. S6) and meanwhile the increase in resistance ofThe mechanism of the correlation between the phenotype and photosynthesis in many lesion mimic mutants, and then the similar light-dependent disease resistance signaling pathways which may be activated in these mutants remain unclear. Therefore, the relationship between photosynthesis and disease resistance needs further study.

Methods

Rice materials

Rice (ssp.cv. Zhejing 22), which is susceptibleto, was acquired from the collection of the Institute of Virologyand Biotechnology, Zhejiang Academy of Agricultural Sciences (ZAAS), Hangzhou, China. The mutant ricederived by EMS mutagenesis of ZJ22wasprovided by the Crop and Nuclear Technology Institute, ZAAS. The rice plants were grown at 28 oC/25 oC (16 h light / 8 h dark), with flux density of 600–800 μmol/(m2?s) and relative humidity of 60%–80%.

Pathogen inoculation and resistant identification

strains were used to inoculate rice leaves using the leaf-clipping method (Kauffman et al, 1973). Briefly, bacterial inoculum was cultured on potato semisynthetic agar (PSA) for 48 h. Bacterial suspension was prepared with a certain concentration (OD600=0.5–1.0). A pair of scissors was dipped into the inoculum and used to clip about 1 cm from the tip of the leaves. At least 16 leaves from 4 plants of eachand ZJ22 were inoculated with the bacteria. After about three weeks, the lengths of bacterial lesions were measured to evaluate resistance. Rice leaves were inoculated withby the spraying method (Zhang et al, 2011). Conidia were resuspended to a concentration of 1×105spores/mLin distilled water. Each of ZJ22 andplants (one-month-old) was sprayed with 5 mL of conidial suspension. Inoculated plants were kept in a small plastic shelter at 25 oCwith 90% humidity underdark treatment for 24 h then followed by a 12 h light/12 h dark cycle. Lesion formation was observed daily. Photographs were taken at7 d after inoculation. Rating scale for different lesion types was measured according to Table S3 (Anonymous, 2002).

TBand DAB staining of leaves

TB staining was used to detect cell death, based on the methods described by Bowling et al (1997) with adjustment for the specific conditions of materials used in this study. The leaves of ZJ22 andwithout lesions were covered with a piece of 2 cm aluminum foil for 10 d. Leaf pieces were dipped in the lactic acid-phenol-trypan blue solution and H2O in a beaker. Boiled for 10 min until dark blue spots had appeared completely. Then, leaf pieces were decolorized in chloral hydrate and incubated at room temperature for 24–48 h. Made frozen slices and observed in a microscope using a 60× oil objective.

DAB staining to determine the sites of H2O2accumulation was done using the methods described by Thordal-Christensen et al (1997). Leaves were shading as described above. Decolorize the leaves and placed into fresh 90% ethanol and cooled to room temperature. They were then freeze-sectioned and examined by microscopy as described above for TB staining.

Determination of chlorophyll content, photosynthetic efficiency and activity of antioxidant enzymes

The rice plants were cultivated to five-leaf stage. The first leaf from the top (Leaf A, with no lesion mimic), the second leaf from the top (Leaf B, with mild lesion mimic) and the third leaf from the top (Leaf C, with severe lesion mimic) were used for determination of chlorophyll content and activity of antioxidant enzyme. The chlorophyll content was detected according to the method of Arnon (1949). Photosynthetic parameters were monitoredwith a portable photosynthesis system (LI-6400, LI-COR, USA). All the photosynthetic measurements were taken at a constant airflow rate of 500 μmol/s and at saturation irradiance with incident photosynthetic photon flux density of 1000μmol/(m2?s). The concentration of ambient CO2was about 385 cm3/m3and the temperature was about 25 oC.APX and SOD activities were detected according to the previous method (Giannopolitis and Ries, 1977; Nakano and Asada, 1981).

qRT-PCR

Total RNA was extracted from different leaf positions using Trizol according to the instructions. Genomic DNA was removed by RNase-free DNase treatment. Total RNA was reverse-transcribed by using the iScript cDNA Synthesis Kit (BIO-RAD, Hercules, USA). qRT-PCR was performed using Ssofast EvaGreen Supermix (BIO-RAD, Hercules, USA) with Light cycler 480 (Roche, Basel, Switzerland). The conditions were as follows: 45 cycles, 95 oC for 30 s, 60 oC for 45 s, and 72 oC for 30 s. House-keeping genewas used as an internal control. Three repeats were performed for each sample. Five PRs,including,,,and,were detected. And DEPs include 14-3-3 proteins, OSAPX2, SOD, OsPR10 and AAA type ATPase were also detected by qRT-PCR. All primers were designed by using Primer Premier 5 and are listed in Table S4.

Extraction of total DNA and gene mapping

We constructed backcross population of wild type ZJ22 and. Total genomic DNA was extracted from mix pool with 30 fresh leaves of F2separation group. The whole genome resequencing of wild type plants andwere performed by Beijing Genomics Institute. SNP detection and analysis were carried out.

Extraction and purification of total proteins

At the five-leaf stage, 2.5 g each from leaves A, B and C were sampled with four replicates for each kind of leaf. The total protein was extracted by the phenol-methanol method (Deng et al, 2007) and then purified using the 2D Clean Up Kit (GE healthcare, UK).

Protein cydye labeling and 2D-DIGE

The purified protein was quantified using the 2D Quant Kit (GE Healthcare, UK) according to the instructions. The absorbance of the sample and the standard solution at 480 nm was measured by a spectrophotometer (Ultrospec1100 pro UV) and the concentration of the test protein was calculated according to the standard curve. Samples (10 μL) containing 50 μg protein were mixed with Cy dyes in the dark, and then adding 1μL Cy3/Cy5 fluorescent dye working liquid, vortexing, centrifuging briefly and placing on ice for 30 min. About 1μL of 10 mmol/L lysine was added to each sample and the mixture was vortexed, centrifuged briefly and placed on ice for 10 min. Finally, About 3 μL Cy2 fluorescent dye solution was added and the mixture was vortexed, centrifuged briefly and placed on ice for 30 min. 2D-DIGE was done according to the method of Dong et al (2017).

Image scan and data analysis

The gel was scanned using the Typhoon 8600 Scanner (GE Healthcare, UK). The region of high protein abundance in the gel was scanned first at 100 μm, and then the entire surface was scanned. The maximum pixel intensity was between 30000–55000. The scanner was set up as follows: the wavelength of the laser scanned on the Cy2 image was 480/430 nm, and the wavelength of the band-pass emission filter was 530/540 nm; the wavelength of the laser scanned on the Cy3 image was 540/525 nm, and the wavelength of the band-pass emission filter was 595/525 nm; the wavelength of the laser scanned on Cy5 image was 635/630 nm, and the wavelength of the band-pass emission filter is 680/630 nm.

Software DeCyder 2D 7.0 (GE Healthcare, UK) was used to analyze the scanned 2D-DIGE image. After the images were imported into the Image Loader software, Differential In-gel Analysis and Biological Variation Analysis were used to detect spots. The limit was set to 1.5-fold change by the Student’s-test (<0.05). Boolean analysis sets were created between the statistic sets and the quantitative or qualitative sets. The spots were compared among four replicates. Only a spot displaying a reproducible change pattern was considered to be a DEP.

MS analysis and database searching

DEPs were obtained by gel electrophoresis except that the loading quantity of the sample was 500 μg, and no fluorescent dyes were used. After electrophoresis, the gel was stained with coomassie brilliant blue (Wang et al, 2012). The 2-DE image was scanned by the UMAX Power Look 2100XL scanner (Maximum Tech, Taiwan, China) at 300 dpi. These images were compared with the 2D-DIGE images to identify spots of interest. Protein spots were manually excised from the gels and cut into small pieces.

Protein samples were digested according to the method of Yan et al (2005) and then subjected to MS analysis, and MS/MS data was analyzed by using a 4800 Plus MALDI TOF/TOFTMAnalyzer (Applied Biosystems, CA, USA). The analytical method was conducted as per Dong et al (2017). Oryza_combine (91081 seqs) was used in protein identification.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

Fig. S1. Genotype frequency profile of progeny on chromosome 2.

Fig. S2. Numbers of differentially expressed protein spots onin comparison with wild type plants.

Fig. S3. Gene ontology classification of differentially expressed proteins.

Fig. S4. Kyoto Encyclopedia of Genes and Genomes enrichment of differentially expressed proteins.

Fig. S5. Differentially expressed proteins in major metabolic pathways of rice.

Fig. S6. Chorophyll content, antioxidase activity and photo- synthetic parameters.

Table S1. Unique protein spots identified by tandem mass spectrometric tandem mass spectrometric.

Table S2. Differentially expressed protein spots identified at three different leaves in wild type plant and.

Table S3. Grade description for screening against.

Table S4. List of primers used for quantitative real-time PCR.

ACKNOWLEDGEMENTS

This study was supported by the Zhejiang Provincial Key Research and Development Plan (Grant No. 2019C02006), the National Key Research and Development Program of China (Grant Nos. 2016YFD0200804 and 2016YFD0100601-15), the Key Program of Zhejiang Provincial Foundation for Natural Science (Grant No. LZ16C130002), the Zhejiang Fundamental Public Welfare Research Program (Grant No. LGN19C140008), and the State Key Laboratory for Managing Biotic and Chemical Threats to Quality and Safety of Agro-products (Grant No. 2010DS700124-ZZ1907).The authors thank Professor M. J. Adams for critically reading the manuscript.

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19 August 2020;

2 March 2021

Mei Qiong (meiqiong@syau.edu.cn); Chen Jianping (jpchen2001@126.com)

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