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First published online March 9, 2007; 10.1104/pp.107.095653 Plant Physiology 144:524-533 (2007) © 2007 American Society of Plant Biologists
Magnaporthe grisea Infection Triggers RNA Variation and Antisense Transcript Expression in Rice1,[W]Department of Plant Pathology (M.G., R.-C.V., H.L., C.J., G.-L.W.) and Ohio Supercomputer Center (E.S.), Ohio State University, Columbus, Ohio 43212; Arizona Genomics Computational Laboratory, BIO5 Institute (V.P., C.S.) and Arizona Genomics Institute and Department of Plant Sciences (H.K., R.W.), University of Arizona, Tucson, Arizona 85721; Fungal Genomics Laboratory, Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695 (R.A.D.); and Rice Genomics Laboratory, Hunan Agricultural University, Changsha, Hunan 410128, China (G.-L.W.)
Rice blast disease, caused by the fungal pathogen Magnaporthe grisea, is an excellent model system to study plant-fungal interactions and host defense responses. In this study, comprehensive analysis of the rice (Oryza sativa) transcriptome after M. grisea infection was conducted using robust-long serial analysis of gene expression. A total of 83,382 distinct 21-bp robust-long serial analysis of gene expression tags were identified from 627,262 individual tags isolated from the resistant (R), susceptible (S), and control (C) libraries. Sequence analysis revealed that the tags in the R and S libraries had a significant reduced matching rate to the rice genomic and expressed sequences in comparison to the C library. The high level of one-nucleotide mismatches of the R and S library tags was due to nucleotide conversions. The A-to-G and U-to-C nucleotide conversions were the most predominant types, which were induced in the M. grisea-infected plants. Reverse transcription-polymerase chain reaction analysis showed that expression of the adenine deaminase and cytidine deaminase genes was highly induced after inoculation. In addition, many antisense transcripts were induced in infected plants and expression of four antisense transcripts was confirmed by strand-specific reverse transcription-polymerase chain reaction. These results demonstrate that there is a series of dynamic and complex transcript modifications and changes in the rice transcriptome at the M. grisea early infection stages.
Rice blast disease, caused by Magnaporthe grisea, is one of the most devastating rice diseases in the world. The rice and M. grisea interaction is becoming a model system for studying plant-fungal interactions due to public availability of the host (Goff et al., 2002  ; Yu et al., 2002; International Rice Genome Sequencing Project, 2005) and pathogen (Dean et al., 2005) genome sequences and the functional genomic resources (http://www.mgosdb.org; Gowda et al., 2006c). The easy genetic manipulation of both organisms has streamlined the functional analysis of the rice and fungal genes that are involved in the defense or pathogenesis. In the last years, several techniques, such as differential display, suppression subtractive hybridization (SSH), and EST were applied to reveal the events at the transcriptome level during rice (Oryza sativa) and M. grisea interaction. Using the differential display method, Kim et al. (2000) isolated 18 defense-related genes from suspension cells treated with a fungal elicitor prepared from M. grisea. SSH is a rapid and effective method to isolate differentially expressed genes as demonstrated in the studies by Kim et al. (2001, 2005), Xiong et al. (2001), Lu et al. (2004), and Han et al. (2004). However, the high level of sequence redundancy in SSH libraries limits its ability to isolate a large number of defense genes. The EST approach was used by Kim et al. (2001) and Jantasuriyarat et al. (2005) to identify defense transcripts on a large scale. The latter sequenced a total of 68,920 ESTs from eight cDNA libraries and identified 13,570 distinct sequences. Because of the high sequencing cost and relative high sequence redundancy, the EST approach was unable to provide deep coverage of the defense transcriptome in the infected rice plants.
Serial analysis of gene expression (SAGE) is one of the most popular genome-wide transcriptome-profiling methods developed in the last decade (Velculescu et al., 1995 In this study, we constructed three rice RL-SAGE libraries, resistant (R), susceptible (S), and control (C), with RNA isolated from the leaf tissues 24 h after M. grisea inoculation. A large set of distinct tags (83,832) were isolated from the three libraries. Interestingly, the mismatch rate of transcript tags in the infected libraries to the rice genomic and expressed sequences was significantly increased due to nucleotide conversions. An enhanced level of expression of the cytidine deaminase and adenine deaminase genes was observed in the infected rice leaves. In addition, we also identified hundreds of antisense transcripts for rice genes that were also highly expressed in the R and S libraries as compared to the C. Our results provide evidence for the involvement of RNA variation and antisense transcript expression during plant-fungal interactions.
Reduction of Defense Transcriptome Size and Presence of Highly Specific Transcript Tags in the R, S, and C Libraries
RL-SAGE library analysis revealed a significant reduction in the number of distinct tags in the R and S libraries in comparison to the C library. The R library had 28,081 distinct tags (11.3% of 248,278 total tags), and the S library had 31,025 distinct tags (10.9% of 282,420 total tags) as compared to 36,034 distinct tags (36.4% of 99,031 total tags) in the C library (Table I
; Fig. 1A
). The distinct tags in each library were further classified into three groups: highly expressed (
A tag with two or more copies was considered a technically significant tag, and a tag with only a singleton was a technically nonsignificant tag. For technically significant tags, an 18.4% reduction in the R library and a 23.7% reduction in the S library were observed as compared to that of the C library (Fig. 1A). About two-thirds of the distinct tags in the three libraries were singleton tags (Fig. 1A). The percentage of singleton tags is similar to that reported in previous SAGE studies (Velculescu et al., 1999 ; Chen et al., 2002). We also defined a tag as a biologically significant tag if it commonly occurred in two or more libraries, and a nonbiologically significant tag if it occurred only in one library. The percent of biologically significant tags present in all three libraries (C, R, and S) was only 3.0% as compared to 7.2% in the R and S libraries, 7.5% in the C and R libraries, and 9.3% in the C and S libraries (Table II ). Even when technically significant tags among the three libraries were used for comparison analysis, only 19.6% to 24.6% commonly occurred in any of the two libraries (data not shown). Intriguingly, a large portion of the tags (76.7%–78.5%) was specifically expressed either in the R, S, or C library (Table II). These results strongly suggest that the R and S transcriptomes might have undergone dynamic and different transcript reprogramming in rice plants at the early infection stages.
To identify whether any fungal genes were expressed in the M. grisea-infected leaf tissues, the RL-SAGE tags from the R and S libraries were matched to the M. grisea genome and annotated gene sequences. Only 24 tags matched the M. grisea genome, but not the rice sequences (Supplemental Table S1). Among the seven tags from the R library, three matched the annotated genes (MGG_12892.5, MGG_08575.5, and MGG_03245.5). Among the 17 tags from the S library, three matched the annotated genes (MGG_06927.5, MMGa04587, and MMGa15070).
When the distinct tags of each library matched the rice reference sequence (RefSeq), the tags in the C library had the highest matching rate (53.0% to genomic DNA and 38.3% to ESTs). On the contrary, only a small fraction of the tags from the R library (17.6% to genomic DNA and 14.5% to ESTs) and the S library (21.0% to genomic DNA and 17.0% to ESTs) matched the rice RefSeq. The matching rate of the technically significant tags (greater than two copies) in the C, R, and S libraries was 70.4%, 67.0%, and 66.6%, respectively, when matched with the ESTs, and was 88.1%, 84.1%, and 85.6%, respectively, when matched with the rice genomic sequence (Fig. 1B). However, the matching rate of singletons in the R and S libraries was much lower than that in the C library (Fig. 1C). Specifically, the matching rate of singletons in the C library was 23.1% to the ESTs and 37.8% to the genomic sequence. Surprisingly, only a very small fraction of the singletons in the R and S libraries matched the ESTs (2.6% in R and 1.5% in S) and the genomic sequence (3.4% in R and 2.0% in S), compared to the C library (Fig. 1C). Similarly, biologically significant common tags and library specific tags were analyzed for sequence matching to the rice RefSeq (Table II). The matching rate of the common tags between the R and S libraries was significantly low (53.7% and 55.7% with the ESTs and genomic sequences, respectively) as compared to that of the common tags of the C and R libraries (74.2% and 77.5%), the common tags of the C and S libraries (75.1% and 79.3%), and the common tags of the C, R, and S libraries (79.4% and 81.6%). A considerable number of specific tags in the C library matched to ESTs (38.5%) and genomic DNA sequences (46.2%). On the contrary, only a very small fraction of the R- and S-specific tags matched the ESTs (4.5% in R and 6.0% in S) and the genomic sequences (5.6% in R and 7.5% in S). These results strongly suggest that the tag sequences in the R and S libraries might have been modified or changed considerably during rice and M. grisea interaction.
To investigate the cause of the mismatch of the transcript tags in the infected plants, one- and two-nucleotide mismatches to the rice RefSeq were allowed in the matching analysis. The tags with a one-nucleotide mismatch were 3,619 (10.0%) in the C library, 6,228 (22.2%) in the R library, and 6,726 (21.7%) in the S library. The number of significant tags with a one-nucleotide mismatch in the three libraries was small (485 in C [1.3%], 358 in R [1.3%], and 372 in S [1.2%]). Many of the one-nucleotide mismatch singleton tags hit to the same genes in the R and S libraries, suggesting that multiple editing events occurred within the same 21-bp region of a gene. Interestingly, the one-nucleotide mismatch rate of the singletons in the R and S libraries to rice annotated genes was 3 times higher than that in the C library (Fig. 3A ; C, 8.7%; R, 20.8%; S, 20.5%). However, the two-nucleotide mismatch analysis did not reveal any significant difference among the tags from the three libraries (Fig. 3A). This indicates that single-nucleotide conversions are the most predominant type of RNA variations in M. grisea-infected plants, although some sequence deletions or additions were also observed (data not shown).
Detailed analysis of one-nucleotide mismatched tags revealed 12 types of nucleotide conversions (Fig. 3B). The A-to-G and U-to-C types were most predominant in the R and S libraries. The gene with the highest numbers of single-nucleotide variations was the Rubisco activase gene (Fig. 4 ), of which 70% of the conversions were found from high-quality sequences (Supplemental Fig. S1). To validate single-nucleotide conversions, reverse transcription (RT)-PCR analysis was performed using independently isolated RNA from C, R, and S reactions. The RT-PCR products were cloned and about 30 RT-PCR clones were sequenced from each library. In the sequenced clones from the R and S libraries, the A-to-G and U-to-C conversions in the Rubisco activase transcripts were confirmed (Supplemental Fig. S2). It is noteworthy that several edited sites were also identified for many pathogenesis-related (PR) protein-encoding transcripts such as chitinase, peroxidase, nonspecific lipid-transfer protein 2, germin protein type 1, or catalase (Supplemental Table S2).
In the rice genome, we identified eight cytidine deaminase genes and four adenine deaminase genes (Supplemental Table S3). However, only LOC_Os07g14150 (cytidine deaminase) and LOC_Os05g28180 (adenine deaminase) were expressed in all three libraries, but with a much higher expression level in the R and S libraries (Fig. 5 ). An independent RT-PCR confirmation experiment showed that the adenine deaminase and cytidine deaminase genes were indeed up-regulated in the infected plants (Fig. 5). These results suggest that transcript sequence variation is associated with RNA-editing pathways in rice upon M. grisea infection.
Induction of Antisense Transcripts in the R and S Transcriptomes
Antisense transcripts in eukaryotes have recently been recognized as important RNA molecules that play vital roles in various cellular pathways (Kumar and Carmichael, 1998
Interestingly, most of the antisense tags (62.2%) from the three libraries perfectly matched to the 5' region of the KOME sense FL-cDNA sequences (Fig. 2), which contradicts a previous report stating that most of the antisense transcripts from the FL-cDNA libraries overlap with the 3' region of the sense transcripts in rice (Osato et al., 2003
We also found that the percentage of antisense tags with two or more copies was much higher in the R (81.2%) and S (89.5%) libraries as compared to the C library (41.2%; Supplemental Fig. S3). Similar to the sense transcript tags, there were much less perfectly matched antisense transcript tags to the rice FL-cDNAs from the R and S libraries as compared to the C library (R, 430 [1.5%]; S, 579 [1.9%]; C, 2,627 [7.3%]). Sequence analysis also revealed that the matching rate of the antisense transcripts in the R and S libraries to the rice RefSeq was increased dramatically when one-nucleotide mismatch was allowed (data not shown). Interestingly, induction or repression of the antisense transcripts for many PR genes was observed in the R and S libraries (Table III
). To confirm the expression of some antisense transcripts, strand-specific RT-PCR using specific antisense primers of four selected genes was performed. The expression of four antisense transcripts detected by RT-PCR was consistent with the tag frequency in the three libraries (Supplemental Fig. S4). All four antisense tags had matches in the rice antisense database (Osato et al., 2003
In the battle to survive in nature, plants have evolved and developed sophisticated mechanisms to recognize pathogens and defend themselves against infection. Rapid activation of defense-related genes is one of the predominant responses to effectively inhibit pathogen invasion. In this study, the defense transcriptome of rice plants was deeply surveyed using the RL-SAGE approach. Dramatic transcriptome reprogramming in the infected rice plants was found 24 h after M. grisea inoculation. The majority of the distinct transcript tags (approximately 75%) were specifically expressed in the R and S reactions. Less than 3,000 transcript tags were commonly present in all three libraries. In comparison with the results from our EST sequencing project (Jantasuriyarat et al., 2005 ), many more differentially expressed genes were identified in the R and S reactions due to deep profiling of transcripts in the RL-SAGE libraries. From 8,000 to 9,000 cDNA clones, only 2,500 to 3,000 distinct ESTs were identified in each library. On the contrary, about 30,000 distinct transcripts were identified after sequencing about 7,000 RL-SAGE clones. Therefore, RL-SAGE is a much more effective method for in-depth transcriptome analysis (Gowda et al., 2004, 2006c). The differentially expressed genes identified from this study offer a rich genomic resource for further detailed functional analysis of rice defense-related genes against M. grisea infection.
In the matching analysis of the total RL-SAGE tags, we found that the matching rate of the R and S library tags to the rice RefSeq was significantly reduced in comparison to the C library. The matching rate of the significant tags was comparable in the C, R, and S libraries because only a small number of edited tags had two copies or more in the R and S libraries. On the contrary, the matching rate of the singletons in these libraries was extremely low (3%–4%). To identify the cause of the low matching rate, we allowed one- to two-nucleotide mismatches in the sequence analysis, which significantly improved the matching rate to both genomic and expressed sequences. Furthermore, various types of single-nucleotide conversions were identified in the mismatched tags. Sequence analysis of the raw reads showed that over 70% of the nucleotide conversions were from high-quality sequencing regions. Independent RT-PCR confirmation of these variations clearly demonstrated that the mismatches were not originated from sequencing errors. This is consistent with the finding by Chen et al. (2002)
These results prompted us to speculate that mRNA might be edited in the infected plants. RNA editing has been well documented for several mitochondrial and chloroplast genomes of plants. However, no study has been reported on the editing of nuclear transcripts in plants. In contrast, transcripts of many nuclear genes in animals have been shown to be edited by a variety of mechanisms at various developmental stages and under stresses (Athanasiadis et al., 2004
Many antisense transcripts were identified in the M. grisea-infected RL-SAGE libraries in this study. Strand-specific RT-PCR using antisense primers of four genes confirmed their expression pattern in the infected plants. One of the antisense transcripts was the classical defense gene PR1. The function of the induced antisense transcripts in M. grisea-infected plants is not known yet. Previous studies have shown that the sense and antisense transcripts might pair in the cells to form dsRNA molecules, which may be the target for RNA editing, splicing, polyadenylation, transport, translation, and small interfering RNA (siRNA)-based gene silencing (Araya et al., 1998
It is also possible that an alternative pathway might be involved in RNA sequence diversity. It has been reported that various reactive oxygen species (ROS), superoxide anion, hydrogen peroxide, and hydroxyl radical could damage RNA and DNA molecules in living cells under stress conditions. RNA is more susceptible to ROS damage as compared to DNA because RNA molecules are widespread in the cytosol, single stranded, and lack protective proteins and repair mechanisms (Zhang et al., 1999 ; Hofer et al., 2006). It has also been shown that RNA damage by ROS is related to human diseases such as Alzheimer's (Nunomura et al., 1999; Honda et al., 2005) and Parkinson's (Zhang et al., 1999). Rapid ROS production was documented during rice-M. grisea interaction (Pasechnik et al., 1998; Mellersh et al., 2002). Whether the rapid production of ROS is one of the causes for RNA variation observed in this study is unknown and requires further evaluation.
Rice Blast Fungal Inoculation and Tissue Collection For the R and S reactions, the avirulent isolate C9240 (from H. Leung, International Rice Research Institute, Philippines) and the virulent isolate Che86061 (from G. Lu, Fujian, China), respectively, were used to inoculate 21-d-old rice (Oryza sativa) plants (japonica ‘Nipponbare’). Rice plants were grown in a Conviron growth chamber at 80% relative humidity with 12 h of light (500 µmol photons m–2 s–1) at 26°C followed by 12 h of darkness at 20°C. The ascospores at 2 x 105 spores mL–1 in a 0.01% Tween 20 solution were sprayed on rice leaves. C plants were sprayed with the 0.01% Tween 20 solution. The inoculated plants were kept in a sealed plastic container in the dark at 26°C for 24 h. Leaves were harvested 24 h after inoculation and kept in a –80°C freezer until RNA isolation.
Total RNA was isolated from the infected and C leaves using the TRIzol method (Invitrogen). Poly(A+) mRNA was purified using the Oligotex mRNA midi kit (Qiagen) according to the manufacturer's instructions. RL-SAGE libraries were generated using an improved procedure reported by Gowda et al. (2004)
Sequence analysis showed that the ditag size ranged from 38 to 44 bp (data not shown). To avoid sequence errors caused by imprecise cleavage (slippage) by MmeI, only three ditag types (40, 41, and 42 bp) were used for the isolation of 21-bp tags, which accounted for 97% of the ditag types in the analyzed sequences. Second, a ditag must have a CATG site at both ends. Third, any tags with an ambiguous base pair (flagged as N) are discarded. The SAGEspy program developed at the Ohio Supercomputer Center (http://www.osc.edu/research/bioinformatics/sagespy/index.shtml) and analysis tools at the Magnaporthe Grisea Oryza Sativa database (http://www.mgosdb.org/sage; Gowda et al., 2006b
The 21-bp RL-SAGE tags from the C, R, and S libraries matched against several rice RefSeq databases such as TIGR ESTs, release version 16.0 (http://www.tigr.org/tigr-scripts/tgi/T_release.cgi?species=rice), KOME FL-cDNA sequences (14; http://cdna01.dna.affrc.go.jp/cDNA), and a nonredundant TIGR rice genome annotation database, release 4 of the TIGR pseudomolecules (January 12, 2006; http://www.tigr.org/tdb/e2k1/osa1/pseudomolecules/info.shtml). Total tags in each library were normalized to 100,000 for comparative expression analyses. The one- and two-nucleotide mismatch alignment of RL-SAGE tags against the RefSeq databases were customized using a stand-alone local BLAST 2 program with word size 7 and E value = 0.01. A set of SAGE alignment parsing programs were developed and used to parse and extract BLAST outputs for statistical data analysis and reports.
To identify the antisense orientation of RL-SAGE tags for the rice RefSeq, we converted all tags into antisense orientation by a reverse-complementation procedure. The antisense tags from the C, R, and S libraries were independently matched against the rice RefSeq. For validating identified antisense tags from the C, R, and S libraries, we matched antisense RL-SAGE tags against longer antisense rice FL-cDNAs available at the KOME database (Osato et al., 2003
About 1.0 µg of total RNA was treated with DNaseI (Invitrogen) and first-strand cDNA synthesized using the alfalfa mosaic virus reverse transcriptase system (Promega) and oligo(dT) primer (Supplemental Table S5). The 3' region (500 bp) of the Rubisco activase gene was amplified by using 5'-Rubisco activase primer and 3'-RACE primer (Supplemental Table S5). PCR products were purified using a Qiagen kit and cloned into the pGEM-T Easy vector system (Promega). About 30 RT clones were sequenced using the M13 forward primer. RT-PCR analysis of adenine deaminase and cytidine deaminase genes was done using gene-specific primers that are listed in Supplemental Table S5.
To amplify antisense transcripts, the strand-specific RT-PCR method was performed as described previously by Røsok and Sioud (2004 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers GSM34282 to GSM34284.
The following materials are available in the online version of this article.
We thank Brandon Childers, Shankaraganesh Manikantan, and James Hatfield for their assistance in data analysis. We are also grateful to Rose Palumbo and Miguel Vega-Sanchez for their critical reading of this manuscript and to all the lab members for their valuable help and discussion during the course of work. Received January 8, 2007; accepted February 15, 2007; published March 9, 2007.
1 This work was supported by the National Science Foundation Plant Genome Research Program (grant nos. DBI 0115642 and 0321437).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Guo-Liang Wang (wang.620{at}osu.edu).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.095653 * Corresponding author; e-mail wang.620{at}osu.edu; fax 614–292–4455.
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ABSTRACT
RESULTS
10 copies), intermediately expressed (two to nine copies), and lowly expressed (single-copy [singleton]) tags. Tag frequency analyses indicated that the R library had many more highly expressed tags (C, 367 tags; R, 1,076 tags; S, 648 tags), and the S library had a higher number of intermediately expressed tags (C, 3,627; R, 2,937; S, 4,990), whereas the number of singleton tags was almost similar among the three libraries (C, 24,401; R, 22,919; S, 23,664). The reduction of the transcriptome size in the R and S libraries is likely due to the increased tag redundancy of highly expressed transcripts. Overall, a total of 83,382 distinct tags were obtained by clustering all the distinct tags from the three libraries. The distribution of distinct tags on the Knowledge-Based Oryza Molecular Biological Encyclopedia (KOME) full-length (FL)-cDNAs was analyzed and shown in 
