The Cytosolic Acetoacetyl-CoA Thiolase TaAACT1 Is Required for Defense against Fusarium pseudograminearum in Wheat

Abstract

Fusarium pseudograminearum is a major pathogen for the destructive disease Fusarium crown rot (FCR) of wheat (Triticum aestivum). The cytosolic Acetoacetyl-CoA thiolase II (AACT) is the first catalytic enzyme in the mevalonate pathway that biosynthesizes isoprenoids in plants. However, there has been no investigation of wheat cytosolic AACT genes in defense against pathogens including Fusarium pseudograminearum. Herein, we identified a cytosolic AACT-encoding gene from wheat, named TaAACT1, and demonstrated its positively regulatory role in the wheat defense response to F. pseudograminearum. One haplotype of TaAACT1 in analyzed wheat genotypes was associated with wheat resistance to FCR. The TaAACT1 transcript level was elevated after F. pseudograminearum infection, and was higher in FCR-resistant wheat genotypes than in susceptible wheat genotypes. Functional analysis indicated that knock down of TaAACT1 impaired resistance against F. pseudograminearum and reduced the expression of downstream defense genes in wheat. TaAACT1 protein was verified to localize in the cytosol of wheat cells. TaAACT1 and its modulated defense genes were rapidly responsive to exogenous jasmonate treatment. Collectively, TaAACT1 contributes to resistance to F. pseudograminearum through upregulating the expression of defense genes in wheat. This study sheds new light on the molecular mechanisms underlying wheat defense against FCR.

Keywords: Fusarium pseudograminearum; cytosolic acetoacetyl-CoA thiolase; jasmonate; plant defense; wheat (Triticum aestivum).

Figure 1

Genomic structure of the TaAACT1 gene. Black squares indicate exons. Variations in the genomic sequence of TaAACT1 in the test wheat cultivars could be divided into two haplotypes (Hap I and Hap II). The number indicates the nucleotide site.

Figure 2

Spatiotemporal transcript patterns of TaAACT1 in wheat toward infection of F. pseudograminearum strain WHF220. (A) Transcript abundance of TaAACT1 in the middle-resistant wheat cultivar CI12633 stems inoculated with F. pseudograminearum for 1, 2, 3, 4, 5, 6, and 7 d. The gene transcript level at noninoculation (none) is set to 1. (B) Transcript abundance of TaAACT1 in roots, stems, leaves, and sheaths of wheat cultivar CI12633 plants. The transcript level of TaAACT1 in uninoculated roots was set to 1. (C) Transcript abundance of TaAACT1 in five wheat cultivars at 4 d postinoculation with F. pseudograminearum. The TaAACT1 transcript level in highly susceptible wheat cultivar Yangmai 158 was set to 1. DI represents the FCR disease index. R represents resistance and mild resistance to FCR. S represents susceptibility to FCR. TaActin was used as an internal control gene. Statistically significant differences were derived from the results of three independent replications (t-test: ** p < 0.01; * p < 0.05).

Figure 3

Sequence and phylogenetic analyses of TaAACT1 protein and 18 other thiolase proteins from other plant species and fungi. (A) Schematic diagram of two domains of TaAACT1 protein. (B) Sequence of TaAACT1 protein. The orange part indicates Thiolase-N domain. The red part indicates Thiolase-C domain. (C) The bootstrapped phylogenetic tree is constructed using the neighbor-joining phylogeny of MEGA 11.0. The scale represents the branch length, and each node reresents bootstrap values from 1000 replicates. † indicates the target protein; The bar (0.1) indicates 10% dissimilarity and distance scale can display the degree of difference.

Figure 4

(A) Si-Fi software off-target prediction results. (B) Scheme of genomic RNAs of the BSMV and the recombinant virus construct BSMV: TaAACT1. The antisense orientation of the TaAACT1 insert is indicated by orange box.

Figure 5

TaAACT1 silencing compromises resistance of wheat CI12633 to F. pseudograminearum. (A) RT–PCR analysis of the transcript of BSMV coat protein (CP) in the wheat plants infected by BSMV:GFP or BSMV:TaAACT1 for 14 d. BSMV-infected symptoms were observed on the wheat leaves at 14 d post-transfection with BSMV:GFP or BSMV:TaAACT1. (B) RT-qPCR analysis of the TaAACT1 gene in the wheat plants infected by BSMV:GFP or BSMV:TaAACT1 for 14 d. The relative transcript level of TaAACT1 in BSMV:GFP-infected CI12633 plants was set to 1. TaActin was used as an internal control. (C) FCR symptoms on the stems of BSMV:GFP-infected and TaAACT1-silenced CI12633 plants at 28 d postinoculation (dpi) with F. pseudograminearum strain WHF220. (D) The average lesion sizes of the BSMV:GFP-infected and TaAACT1-silenced plants. (E) The infection types of the BSMV:GFP-infected and TaAACT1-silenced plants in two batches of disease tests ~28 d postinoculation (dpi) with F. pseudograminearum. ** (Student’s t-test, p < 0.01) represents statistically significant differences derived from at least three biological replications. Bars represent standard error of the mean.

Figure 6

Transcript levels of TaChitinase 2 (TaChit2), TaChitinase 3 (TaChit3), TaChitinase 4 (TaChit4), and TaDefensin in BSMV:GFP-infected and TaAACT1-silenced wheat CI12633 plants at 4 dpi with F. pseudograminearum WHF220. Relative transcript abundances of the tested genes (TaChit2, TaChit3, TaChit4, and TaDefensin) in TaAACT1-silenced CI12633 plants were quantified relative to thosein BSMV:GFP-infected CI12633 plants (set to 1). Statistically significant differences between TaAACT1-silenced and BSMV:GFP-infected CI12633 plants were determined based on three replications using Student’s t-test (t-test: ** p < 0.01). Bars represent standard error of the mean. TaActin was used as internal control.

Figure 7

The transcript levels of TaAACT1 and its regulated defense genes in wheat treated with exogenous MeJA and ABA. (A) Transcript level of TaAACT1 in leaves of wheat cultivar CI12633 after exogenous application of 0.05 mM MeJA. The transcript level of TaAACT1 in wheat plants at mock (H₂O) treatment for 0.5 h is set to 1. (B) Transcript level of TaAACT1 in leaves of wheat cultivar CI12633 after exogenous application of 0.1 mM ABA. The transcript level of TaAACT1 in mock (H₂O)-treated (0.5 h) wheat plants is set to 1. (C) Transcript levels of defense genes including TaChit2, TaChit3, TaChit4, and TaDefensin in wheat cultivar CI12633 leaves after exogenous application of 0.05 mM MeJA. The transcript levels of the tested genes in wheat plants at mock (H₂O) treatment for 0.5 h is set to 1. Statistically significant differences (* p < 0.05, ** p < 0.01) are analyzed based on three replications using Student’ s t-test. Bars indicated the SD of the mean. TaActin was used as internal control.

Figure 8

Subcellular localization of TaAACT1 in wheat mesophyll protoplasts. The fused TaAACT1-GFP protein and control GFP protein are transiently expressed in wheat mesophyll protoplasts. Scale bars = 20 μm. Green color represents the green fluorescence. Red color represents chloroplast.