The branched-chain amino acid aminotransferase TaBCAT1 modulates amino acid metabolism and positively regulates wheat rust susceptibility

Abstract

Plant pathogens suppress defense responses to evade recognition and promote successful colonization. Although identifying the genes essential for pathogen ingress has traditionally relied on screening mutant populations, the post-genomic era provides an opportunity to develop novel approaches that accelerate identification. Here, RNA-seq analysis of 68 pathogen-infected bread wheat (Triticum aestivum) varieties, including three (Oakley, Solstice and Santiago) with variable levels of susceptibility, uncovered a branched-chain amino acid aminotransferase (termed TaBCAT1) as a positive regulator of wheat rust susceptibility. We show that TaBCAT1 is required for yellow and stem rust infection and likely functions in branched-chain amino acid (BCAA) metabolism, as TaBCAT1 disruption mutants had elevated BCAA levels. TaBCAT1 mutants also exhibited increased levels of salicylic acid (SA) and enhanced expression of associated defense genes, indicating that BCAA regulation, via TaBCAT1, has a key role in SA-dependent defense activation. We also identified an association between the levels of BCAAs and resistance to yellow rust infection in wheat. These findings provide insight into SA-mediated defense responses in wheat and highlight the role of BCAA metabolism in the defense response. Furthermore, TaBCAT1 could be manipulated to potentially provide resistance to two of the most economically damaging diseases of wheat worldwide.

Figure 1

RNA-seq analysis can be used to define the wheat variety in Pst-infected wheat samples. A, Pst-infected wheat samples cluster genetically based on variety. Phylogenetic analysis was carried out using 96 Pst-infected wheat samples with a maximum-likelihood model (227,267,910 nucleotide sites). Scale bar represents nucleotide substitutions per site; colors reflect wheat variety; stars indicate samples from a wheat cultivar with a single sample. Bootstrap values are provided in Supplemental Data File 2. B, The three wheat varieties Oakley, Solstice and Santiago have differing levels of susceptibility to the dominant Pst pathotypes (races) in Europe. Each of the three varieties was subjected to Pst infection with two isolates (F22 and 13/14) and infection types (IT) recorded 12 dpi following the 0–4 scale (McIntosh et al., 1995). Values represent an average from five independent plants (Supplemental Table 1). C, Principal component (PC) analysis of wheat gene expression profiles illustrates that samples group together by wheat variety, with two well-defined groups: (1) Solstice and (2) Oakley and Santiago (blue, green, and black font, respectively)

Figure 2

Wheat varieties with different levels of Pst susceptibility display variation in expression of genes linked to defense-related processes and amino acid metabolism. A, Gene expression profiling of Pst-infected samples identified a total of 61,700 differentially expressed transcripts that were assigned to 12 co-expression clusters (I–XII) that displayed similar patterns of differential expression between the three wheat varieties Oakley, Solstice and Santiago. B, A number of defense-related processes defined in GO terms were enriched across gene expression clusters. C, Processes linked to amino acid metabolism were also enriched across expression clusters. Enrichment was determined based on Fisher’s exact test and GO terms were considered enriched in one cluster at p < 0.0001

Figure 3

TaBCAT1 expression is elevated early during infection with Pst and Blumeria graminis f. sp. tritici (Bgt). A, A total of 37 transcripts annotated with GO terms linked to BCAA biosynthetic processes were identified across co-expression clusters, with the highest enrichment in cluster VII, followed by cluster XII. B, Gene expression analysis of wheat samples infected with Pst and Blumeria graminis f. sp. tritici (Bgt) revealed three gene homeologs (red highlight) uniquely upregulated at 24 hpi and 48 hpi, respectively. C, These three gene homeologs (TaBCAT1-A, TaBCAT1-B, and TaBCAT-D) were upregulated early during infection with Pst and Bgt and also during abiotic stress. Expression levels for the 37 transcripts were assessed in publicly available wheat RNA-seq data. mo, mock; fu, Fusarium; st, Septoria tritici; pm, powdery mildew (Bgt); sr, stripe rust (Pst); hs, heat stress; ds, drought stress; h, hour; d, day; n, number of replicates. Error bars represent standard errors

Figure 4

TaBCAT1 disruption mutants display reduced susceptibility to Pst. A, TaBCAT1-A^Q50*, TaBCAT1-B^R366-, and TaBCAT1-A^Q50* TaBCAT1-B^R366- disruption mutants all displayed limited sporulation, higher degrees of necrosis and less chlorosis when infected with Pst isolate 13/14 and compared with the Kronos wild-type (WT). Negative controls included Kronos WT and Kronos ethyl methanesulfonate mutants (Kronos860 and K2898xK860) carrying WT alleles of TaBCAT1. Images were captured 20 dpi. B, The percentage of leaf infection was significantly reduced in TaBCAT1 disruption mutants at 20 dpi. Asterisks denote statistically significant differences between each pair of conditions (***p < 0.001, **p < 0.01, two-tailed t-test). Bars represent median values, boxes signify the upper (Q3) and lower (Q1) quartiles, and whiskers are located at 1.5 the interquartile range

Figure 5

TaBCAT1 expression early during Pst infection is required for susceptibility. A, A controlled time-course of infection was carried out with Pst isolate F22 and wheat varieties Oakley, Solstice and Santiago. S, urediniospore; SV, sub-stomatal vesicle; IH, invasive hyphae; HM, haustorial mother cell; H, haustorium; P, pustule; G, guard cell. B, During Pst infection, TaBCAT1 expression at 12 hpi was highest in the most susceptible variety Oakley, whereas the most resistant variety Santiago displayed a significant reduction in TaBCAT1 expression (highlighted area). Two independent leaves from the same plant were pooled and three independent plants analyzed for TaBCAT1 expression by RT-qPCR at 12 hpi, 2 dpi, 5 dpi, 9 dpi, and 11 dpi. TaBCAT1 expression was compared between Pst-infected and mock-inoculated plants for each time point per variety. Asterisks denote statistically significant differences (***p < 0.005, **p < 0.01, *p < 0.05; two-tailed t test). Error bars represent standard deviations. C, D, Histological studies using a fungus-specific fluorescent dye revealed differences in the extension of internal fungal structures between WT and the TaBCAT1-A^Q50* TaBCAT1-B^R366- disruption mutant. The number of germinating spores assessed was as follows: Kronos WT n = 229, TaBCAT1-A^Q50*n = 114, TaBCAT1-B^R366-n = 297, TaBCAT1-A^Q50* TaBCAT1-B^R366-n = 152. The number of internal structures measured was as follows: Kronos WT n = 9, TaBCAT1-A^Q50*n = 2, TaBCAT1-B^R366-n = 10, TaBCAT1-A^Q50* TaBCAT1-B^R366-n = 3. Letters indicate significant differences determined using Duncan’s multi range test (p < 0.05). Bars represent median values, boxes signify the upper (Q3) and lower (Q1) quartiles, and whiskers are located at 1.5 the interquartile range. E, WT plants showed clear Pst hyphal development at 4 dpi. F, Hyphal growth was not observed for TaBCAT1-A^Q50* TaBCAT1-B^R366- at 4 dpi. G, At 6 dpi WT plants showed large, intricate fungal structures. H. The TaBCAT1-A^Q50* TaBCAT1-B^R366- disruption mutant at 6 dpi showed a unique phenotype where germinating spores reached stomata and entered the underlying sub-stomatal space, but further fungal growth was absent. Scale bars in E–H represent 100 µm; S, urediniospore; GT, germ tube; SSV, sub-stomatal vesicle; IH, invasive hyphae

Figure 6

PR gene expression and free SA levels are enhanced following disruption of TaBCAT1 in the absence of infection A. The relative expression of five of the six PR genes tested was significantly upregulated in TaBCAT1-A^Q50*, TaBCAT1-B^R366-, and/or TaBCAT1-A^Q50* TaBCAT1-B^R366- disruption mutants in the absence of rust infection. Letters indicate significant differences determined using Duncan’s multi range test (p < 0.05). Kronos WT n = 3, TaBCAT1-A^Q50*n = 3, TaBCAT1-B^R366-n = 3-4, TaBCAT1-A^Q50* TaBCAT1-B^R366-n = 4. B, This was accompanied by a two-fold increase in free SA levels in the TaBCAT1-A^Q50* TaBCAT1-B^R366- disruption mutant line in the absence of rust infection. Kronos WT n = 5, TaBCAT1-A^Q50* TaBCAT1-B^R366-n = 4. C, The levels of TaPR1, TaPR2, TaPR3, TaPR4, and TaPR9 expression were also significantly higher in TaBCAT1-silenced plants. Expression levels were assessed 9 dpvi using RT-qPCR in plants silenced with BSMV::TaBCAT1 (n = 4) and compared to the negative control, where BSMV::msc4D (n = 4) was utilized as a viral infection control. Asterisks denote statistically significant differences between each pair of conditions (**p < 0.01, *p < 0.05; two-tailed t test). Bars represent median values, boxes signify the upper (Q3) and lower (Q1) quartiles, and whiskers are located at 1.5 the interquartile range

Figure 7

TaBCAT1 localizes to the mitochondria where it may regulate BCAA levels that correspond to Pst susceptibility during infection. A, TaBCAT1-A co-localized with the mitochondrial marker ScCOX4. TaBCAT1:GFP and ScCOX4:RFP were transiently co-expressed in N. benthamiana and images captured 2-day post-infiltration. White arrowheads highlight overlapping GFP and RFP signals in mitochondria. Images are representative of >10 images captured, which all displayed co-localization of TaBCAT1:GFP and ScCOX4:RFP. Left panels, individual TaBCAT1:GFP (top) and ScCOX4:RFP (bottom) localization patterns, right panel, TaBCAT1:GFP and ScCOX4:RFP merged image illustrating co-localization. Scale bar represents 10 µm. B, Several BCAAs and Asp-derived amino acids were enhanced in TaBCAT1-A^Q50* TaBCAT1-B^R366- (n = 4) compared to Kronos wild type (n = 5). Val, Thr, and Lys displayed a significant increase in the double mutant. C, The level of the BCAAs Val, Leu, and Ile and the Asp-derived amino acid Thr were significantly increased at 24 hpi in the resistant variety Santiago (n = 5) during Pst infection (isolate F22) when compared to the susceptible variety Oakley (n = 5). The level of the BCAAs and Asp-derived amino acids were assessed at 12, 24, and 48 hpi. Asterisks denote statistically significant differences (***p < 0.001, *p < 0.05; two-tailed t test). Bars represent median values, boxes signify the upper (Q3) and lower (Q1) quartiles, and whiskers are located at 1.5 the interquartile range

Figure 8

Levels of the three BCAA-related 2-hydroxyacids are unchanged in the TaBCAT1 disruption mutant, but exogenous application of ILA severely perturbs Pst infection. A, Levels of the three BCAA-related 2-hydroxyacids (ILA, VA, and LA) were determined in the TaBCAT1 disruption mutant and Kronos wild-type plants in the absence of rust infection. Leaves from three independent plants were assessed in each case. B, Exogenous application of ILA 72 or 24 h prior to Pst infection dramatically reduced disease progression. Oakley plants were pre-treated with ILA at 0.5, 1, and 2 mM or with dH₂0 (0-mM ILA) 72 or 24 h prior to inoculation with Pst isolate 13/14 and infection types recorded 15-day post-infection using the 0–4 scale (McIntosh et al., 1995). Values represent an average from 10 plants per treatment, with the first (L1), second (L2), and third (L3) leaves analyzed. C, Histological studies using a fungus-specific fluorescent dye revealed that where internal fungal structures formed, their extension was only moderately reduced following ILA treatment at 0.5, 1, or 2 mM prior to Pst infection. D–G, Images illustrate the moderate reduction in Pst internal fungal structures when plants were treated with ILA 24 h prior to Pst infection. Scale bar represents 100 µm. S, urediniospore; GT, germ tube; SSV, sub-stomatal vesicle; IH, invasive hyphae. For the box plots, the bars represent median values, boxes signify the upper (Q3) and lower (Q1) quartiles, and whiskers are located at 1.5 the interquartile range

Figure 9

Model illustrating the role of TaBCAT1 in the defense response. Disruption of TaBCAT1 (downward blue arrow) leads to an elevation in BCAA levels, free SA accumulation and enhanced expression of pathogenicity-related genes (TaPR1, 2, 4, 3, and 9), stimulating an increase in resistance to Pst and Pgt infection (upward blue arrows). The connection between elevated BCAAs and SA is currently unknown (dotted arrow). In contrast, wild type (WT) expression of TaBCAT1 (upward gray arrow) leads to lower levels of BCAAs, SA, PR gene expression, and resistance to Pst (downward gray arrows)