Identification and characterization of wheat long non-protein coding RNAs responsive to powdery mildew infection and heat stress by using microarray analysis and SBS sequencing

Abstract

Background: Biotic and abiotic stresses, such as powdery mildew infection and high temperature, are important limiting factors for yield and grain quality in wheat production. Emerging evidences suggest that long non-protein coding RNAs (npcRNAs) are developmentally regulated and play roles in development and stress responses of plants. However, identification of long npcRNAs is limited to a few plant species, such as Arabidopsis, rice and maize, no systematic identification of long npcRNAs and their responses to abiotic and biotic stresses is reported in wheat.

Results: In this study, by using computational analysis and experimental approach we identified 125 putative wheat stress responsive long npcRNAs, which are not conserved among plant species. Among them, some were precursors of small RNAs such as microRNAs and siRNAs, two long npcRNAs were identified as signal recognition particle (SRP) 7S RNA variants, and three were characterized as U3 snoRNAs. We found that wheat long npcRNAs showed tissue dependent expression patterns and were responsive to powdery mildew infection and heat stress.

Conclusion: Our results indicated that diverse sets of wheat long npcRNAs were responsive to powdery mildew infection and heat stress, and could function in wheat responses to both biotic and abiotic stresses, which provided a starting point to understand their functions and regulatory mechanisms in the future.

Figure 1

Expression patterns of wheat long npcRNAs TapmlnRNA19 (a) and TapmlnRNA30 (b) in response to powdery mildew inoculation (12hai) as determined by qRT-PCR analysis, S-0H: before Bgt inoculation in susceptible (S) genotype, S-12H: 12 hrs after Bgt inoculation in S genotype, R-0H: before Bgt inoculation in resistant (R) genotype, R-12H: 12 hrs after Bgt inoculation in R genotype.

Figure 2

Expression patterns of wheat long npcRNAs TahlnRNA27 (a) and TalnRNA5 (b) in response to heat stress. CS-0h: before heat stress treatment for heat susceptible genotype Chinese Spring (CS), CS-1h: after 1 hour heat stress treatment, TAM-0h: before heat stress treatment for heat tolerant genotype TAM107 (TAM), TAM-1h: after 1 hour heat stress treatment.

Figure 3

Expression pattern of wheat long npcRNA TalnRNA5 and its corresponding miRNA before or 12hai in both disease resistant genotype (R) and susceptible genotype (S). (a) The expression level of TalnRNA5 as determined by qRT-PCR. (b) The expression pattern of miR2004 based on high throughput sequencing. (c) Northern blot analysis for miR2004 expression before or 12hai in S genotype and R genotype.

Figure 4

The positions of siRNAs matching to the TapmlnRNA11.

Figure 5

Expression patterns of wheat long npcRNAs and their corresponding siRNAs before or 12hai in S genotype and R genotype. (a) The expression pattern of TapmlnRNA11 in wheat microarray analysis. (b) The abundance of corresponding siRNAs matching TapmlnRNA11 based on high-throughput sequencing.

Figure 6

The expression pattern of TalnRNA21 in response to powdery mildew inoculation (a) and heat stress (b) based on microarray analysis.

Figure 7

The expression patterns of TalnRNA9 in response to powdery mildew inoculation (a) and heat stress (b) as determined by qRT-PCR.

Figure 8

The expression patterns of TahlnRNA12, TahlnRNA23 and TahlnRNA29 1 h after heat stress in heat sensitive genotype ('CS') and heat tolerant genotype ('TAM107') based on microarray analysis.

Figure 9

The H3K9 acetylation levels of TalnRNA5 and TapmlnRNA19 in S and R genotypes before or 12 hrs after powdery mildew inoculation as determined by qRT-PCR.

Figure 10

Expression patterns of sense and antisense sequences for TalnRNA9 and TalnRNA12 before or 12 hrs after Bgt inoculation in S genotype and R genotype. (a) Expression patterns of sense sequences revealed by microarray analysis. (b) Expression patterns of antisense sequences as determined by qRT-PCR.

Figure 11

Expression patterns of TalnRNA5, TapmlnRNA19, TalnRNA9 and TapmlnRNA30 in eight tissues as determined by qRT-PCR.

Figure 12

The 15 short possible open reading frames (ORFs) positioned in TapmlnRNA26.

Figure 13

Schematic representation of computational method for long npcRNA identification.