Magnaporthe grisea infection triggers RNA variation and antisense transcript expression in rice

Abstract

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.

Figure 1.

The distinct RL-SAGE tags of the C, R, and S libraries and their hits in the rice genomic and TIGR EST sequences. A, Number of total distinct, significant (greater than two copies), and singleton tags in the three libraries. B, Matching results of the significant tags to both genomic and EST sequences. C, Matching results of the singletons to both genomic and EST sequences.

Figure 2.

Location of sense and antisense RL-SAGE tags on the rice FL-cDNAs. Each FL-cDNA was equally divided into three portions (3′, mid, and 5′). The distinct sense and antisense tags from the three RL-SAGE libraries were mapped to the FL-cDNAs using the SAGEspy program (Gowda et al., 2006b). The number of hits and the percent of tags are shown on the top of each category.

Figure 3.

Mismatch analysis of RL-SAGE tags against annotated rice genes. Total distinct tags (A) from the C, R, and S libraries were matched against TIGR annotated rice genes by allowing one and two mismatches. The percent on the top of each bar represents the proportion of the matched tags in each library. Among the one-mismatch tags, the number of tags with specific nucleotide substitutions in C, R, and S libraries is shown in B.

Figure 4.

Nucleotide conversions in Rubisco activase transcripts. Nucleotide conversions are shown with bold and underlined letters. The direction (5′–3′) of the transcripts is shown using an arrow. The copy number of the tags in each library is shown at the left side (C198, R217, S342 denotes 198 tags in the C library, 217 tags in the R library, and 342 tags in the S library). The bold and italicized letters are nucleotide conversions confirmed by sequencing of the RT-PCR fragments (the sixth and ninth tags of Transcript 1). The box at the right-top corner is showing the antisense ESTs for Rubisco activase gene.

Figure 5.

RT-PCR analysis of the adenine deaminase and cytidine deaminase genes. Tag frequency in each library is shown in the box above the gel picture. The ubiquitin gene was used as the expression C for RT-PCR amplification.

Figure 6.

A working model for RNA sequence diversity during rice and M. grisea interaction. Once an M. grisea conidium lands on the rice leaf surface, an infectious structure, called appressorium, develops at the tip of the germ tube. The highly melanized appressorium generates enormous turgor pressure and penetrates through the rice epidermal cells within 24 h of inoculation (Talbot, 1995). Through the penetration peg, the fungus may secrete various proteins (Ellis et al., 2005), mobile elements (Vaughn et al., 1995), and even free radicals into host cells. Fungal secretory molecules may interact with host factors leading to the synthesis of sense and antisense transcripts for defense-related genes. Then both sense and antisense transcripts may pair to form dsRNA in the nucleus. dsRNA may be targeted by the RNA-editing complex, including cytidine deaminase and adenine deaminase (Nie et al., 2006). Edited dsRNAs might be retained in the nucleus and degraded, generating miRNAs and siRNAs (Blow et al., 2006). Translation of edited transcripts may lead to production of protein diversity (Li et al., 2006). miRNAs and siRNAs may affect translation, transcription, and DNA replication (Li et al., 2006) in the nucleus. RNA variation in host transcripts could affect the outcome of the rice and M. grisea interaction: host resistance or susceptibility.