Transcriptome Analyses Of Resistant And Susceptible Wheat Near-isogenic Lines Following Inoculation With Blumeria Graminis F. Sp. Tritici

Powdery mildew, caused by Blumeria graminis f. sp. tritici(Bgt), is one of the most important diseases of wheat(Triticum aestivum L.). In 1980 s, twice epidemics of wheat powdery mildew were occurred in China. Since then, their occurrence areas were on a higher level, and had become one of the major limiting factors of wheat high and stable yields. The wheat powdery mildew fungus could mutate and become a new physiological race in a relatively short time and the cultivar resistance to powdery mildew, containing a single resistance gene, could decrease or even lose in the short term. Therefore, understanding the signaling pathways and plant responses after inoculation will help scientists uncover the molecular mechanisms of disease resistance and will further the use of resistance genes in cultivar breeding. Transcriptome pattern differences in response to Bgt. infection were investigated in two wheat near-isogenic lines(NILs) that differed for the powdery mildew resistance gene, Pm2 at two timepoints, 16 hpi and 96 hpi. The results are list as follows:(1) Transcriptome sequencing by RNA-seq and de novo assemblyTo identify differential expressed genes in Chancellor, L031, and their F1 hybrid after inoculation with the Bgt. isolate E09, leaf RNA samples of the three lines at two different timepoints(16 and 96 hpi) were prepared and sequenced using the Illumina sequencing platform. The 6 RNA-seq libraries included L031, F1 and Chancellor at 16 hpi(designated as T1, T2 and T3) and long-term inoculation at 96 hpi(designated as T4, T5 and T6). After cleaning and checking the read quality, 4.16 G, 3.62 G and 4.06 G of clean data were generated at 16 hpi, while 4.23 G, 3.47 G, and 4.15 G clean data were generated at 96 hpi, respectively. These reads were then assembled de novo using Trinity platform software, resulting in 316,787 transcripts and 112,033 unigenes. In total, 63,210 unigenes were annotated, and the percentage of aligned reads mapping to unigenes in the library was generally approximately 80%, which indicated an acceptable quality of the aligned reads. Comparing the clean data with a specific reference genome, the efficiency of sample data aligned to the reference genome ranged from 79.68% to 83.01%.(2) Differentially expressed genes between L031, F1 and Chancellor in response to Bgt. E09Based on the RPKM data, putative DEGs were identified using an FDR of less than 0.01 and a fold change greater than 2. Without reference genome, comparison between the L031 and Chancellor libraries at 16 hpi(T1 vs. T3) revealed that 2,932 genes were differentially expressed. Similarly, 1,419 DEGs were identified from the comparison between F1 and Chancellor(T2 vs. T3) at 16 hpi. In the long-term inoculation treatment(96 hpi), 4,697 and 4,583 DEGs were identified between T4 vs. T6 and T5 vs. T6, respectively. When there is reference genome, T1 vs. T3 has 5,052 DEGs, T2 vs. T3 contains 1,832 DEGs. There are 4,710 and 4,507 DEGs found in 96hpi-comparison. It was clear that more DEGs were identified at 96 hpi than at 16 hpi, which indicated that more down-stream genes were trigged in the later stages than in the early stages post-inoculation. The results also confirmed that the earlier stage post-inoculation is the most important timepoint for studying the expression and response of race-specific resistance genes after fungal attack in wheat. In this study, the Venn diagram of the differentially co-regulated genes revealed overlapping DEGs between L031 vs. Chancellor and F1 vs. Chancellor at 16 hpi and at 96 hpi. The overlapping DEGs were found functioning in translation, transcription, signal transduction at 16 hpi, while at 96 hpi the co-regulated DEGs functioned in translation, posttranscriptional modification and energy production and transformation. These overlapping DEGs may play an important role in disease resistance.(3) Genes involved in early and long-term signaling of resistance to Bgt.Comparison between 16 and 96 hpi of the L031 libraries(T1 vs. T4) showed that 2,699/1,958 genes were differentially expressed. Similarly, 1,952/3,456 DEGs were identified from comparison between 16 and 96 hpi of the F1 hybrid(T2 vs. T5), and 4,697/1,499 DEGs were identified in the Chancellor libraries(T3 vs. T6)(without and with a reference genome). The results showed that long-term inoculation by Bgt. triggered more robust alterations in gene expression than those observed a short time after inoculation, thus resulting in more up-regulated genes at 96 hpi. As the leaves of Chancellor were attacked and damaged by Bgt. inoculation, more DEGs were found than in resistant genotypes(L031 and F1). KEGG enrichment analysis also revealed that several pathways were specially found in the later stages post-inoculation, including secondary metabolic pathways, such as “tropane, piperidine and pyridine alkaloid biosynthesis,” “steroid biosynthesis,” “ABC transporters,” and so on.(4) R genes involved in Pm2 response to Bgt. E09We selected those DEGs whose expression levels were zero or close to zero in the susceptible parent while having high RPKM values in L031 and the F1 hybrid at the two timepoints. Some of the DEGs were well-known defense-related genes. In this study, eight transcripts encoding the LRR family of receptor serine/threonine-protein kinases were up-regulated with the onset of Bgt. infestation in the resistant phenotype(T1, T2, T4 and T5) compared to susceptible phenotype(T3 and T6) in both treatments. The RPKM values of five unigenes were much lower(some were 0) in Chancellor, and these five genes might be involved in gene-for-gene mode of resistance and are probably responsible for the perception of invasion and activating down-steam signaling cascades to induce defense responses. 47 overlapping unigenes between T1 vs.T2 vs. T3 and T4 vs. T5 vs. T6 that showed significant down-regulation(RPKM = 0) in Chancellor at two timepoints were identified. These 47 genes correspond to disease resistance proteins RGA, RPM and RPP, a few types of receptor-like protein kinases, and some hypothetical proteins. When aligned to the reference genome, 76 co-regulated new unigenes were found and 19 of these genes were annotated to disease resistance proteins RGA, RPM and RPP, Putative LRR receptor-like serine/threonine-protein kinase, Cysteine-rich receptor-like protein kinase, G-type lectin S-receptor-like serine/threonine-protein kinase, WRKY transcription factor, and so on.