OsWRKY93 Dually Functions Between Leaf Senescence and in Response to Biotic Stress in Rice

Abstract

Cross talking between natural senescence and cell death in response to pathogen attack is an interesting topic; however, its action mechanism is kept open. In this study, 33 OsWRKY genes were obtained by screening with leaf aging procedure through RNA-seq dataset, and 11 of them were confirmed a significant altered expression level in the flag leaves during aging by using the reverse transcript quantitative PCR (RT-qPCR). Among them, the OsWRKY2, OsWRKY14, OsWRKY26, OsWRKY69, and OsWRKY93 members exhibited short-term alteration in transcriptional levels in response to Magnaporthe grisea infection. The CRISPR/Cas9-edited mutants of five genes were developed and confirmed, and a significant sensitivity to M. oryzae infection was observed in CRISPR OsWRKY93-edited lines; on the other hand, a significant resistance to M. oryzae infection was shown in the enhanced expression OsWRKY93 plants compared to mock plants; however, enhanced expression of other four genes have no significant affection. Interestingly, ROS accumulation was also increased in OsWRKY93 enhanced plants after flg22 treatment, compared with the controls, suggesting that OsWRKY93 is involved in PAMP-triggered immune response in rice. It indicated that OsWRKY93 was involved in both flag leaf senescence and in response to fungi attack.

Keywords: OsWRKY93; biotic stress; flag leaf; rice; senescence.

FIGURE 1

Heat map diagram of relative gene expression levels of 33 OsWKRYs from total 102 WRKYs (Supplementary Dataset S1) in rice flag leaves at six stages during aging. Developmental stages comprising six stages of flag leaf (0, 1, 2, 3, 4, and 5 weeks after heading). Expression values were scaled by Log2Fold change ≥ 1 and FDR < 0.05 normalized to 0W stage of flag leaf development. 10 OsWRKY candidates are indicated with yellow highlight.

FIGURE 2

Analyses of several OsWRKYs expression level in rice flag leaves during natural senescence. The expression level was assessed by RT-qPCR. All values were normalized to OsACTIN expression. Box-and-whisker plots show the median value (horizontal lines), interquartile range (boxes), and minimum and maximum values (whiskers). Three biological replicates and three technique replicates were used. The broken-line graphs indicate expression profiles of 11 OsWRKYs from RNA-seq dataset. Asterisks indicate significant differences relative to the 0W controls calculated using the Student t-test: *P < 0.05; **P < 0.01; and ***P < 0.001. The leaf Y_axis denotes relative expression by RT-qPCR. The right y-axis denotes ratio of the fold change of RPKM compared with 0W by RNA-seq. 0W means 0 week after heading.

FIGURE 3

Expression analysis of five OsWRKY genes and the defense-related marker gene OsNAC4 in response to M. oryzae infection. qRT-PCR analysis of five OsWRKYs and OsNAC4 in WT at 0, 24, 48, 72, 96, and 108 h after pathogen treatment. The Y-axis represents the relative expression level normalized to OsACTIN. Box-and-whisker plots show median value (line within box), interquartile range (boxes), and minimum and maximum values (whiskers). Three biological replicates and three technique replicates were used. Asterisk indicate significant differences (**P < 0.01, and ***P < 0.001) based on Student t-test compared to 0 h.

FIGURE 4

Generation and analysis of the OsWRKY93 transgenic lines. (A) Real-time quantitative PCR experiments showing expression changes of OsWRKY93 in Kitaake and the OsWRKY93_VP64. (B) Representative leaves of Kitaake and the OsWRKY93_VP64 3 and 4 days after inoculation with M. oryzae. Pathogen infection assays were performed on three biological replicates. (C) Schematic diagram for the CRISPR-edited mutant of OsWRKY93. Yellow boxes and black lines represent exons and introns, respectively. The sgRNA target is cyan. (D) Sequence of the oswrky93-1 mutant identified from transgenic plants of the OsWRKY93 sgRNA target. The reverse complementary sequence of the PAM sequence (5’-CGG-3’) of the sgRNA target is green. The red T represents a one-base insertion. (E) Representative leaves of Nipponbare and oswrky93-1 3 and 4 days after inoculation with M. oryzae. Pathogen infection assays were performed on three biological replicates.

FIGURE 5

ROS accumulation in rice leaves after flg22 treatment. (A) A flg22-induced ROS burst in the OsWRKY93_VP64 and Kitaake plants. (B) A flg22-induced ROS burst in the oswrky93-1 and Nipponbare plants. Rice leaf disks were treated with 1 μM Flg22 or water. Error bars represents the SE (n = 3–5).

FIGURE 6

Phenotyping of detached leaves after darkness treatment. (A) A delaying leaf senescence shown in the OsWRKY93_VP64 (OsW93_vp64) compared to Kitaake plants. (B) An early leaf senescence shown in the oswrky93-1 compared to Nipponbare (NIP) plants. Detached leaf pieces of rice were incubated with 1 μM MES (pH8.5) buffer after darkness treatment for 0, 48, 72, and 84 h.