In vivo assembly of the sorgoleone biosynthetic pathway and its impact on agroinfiltrated leaves of Nicotiana benthamiana

Abstract

Sorgoleone, a hydrophobic compound exuded from root hair cells of Sorghum spp., accounts for much of the allelopathic activity of the genus. The enzymes involved in the biosynthesis of this compound have been identified and functionally characterized. Here, we report the successful assembly of the biosynthetic pathway and the significant impact of in vivo synthesized sorgoleone on the heterologous host Nicotiana benthamiana. A multigene DNA construct was prepared for the expression of genes required for sorgoleone biosynthesis in planta and deployed in N. benthamiana leaf tissues via Agrobacterium-mediated transient expression. RNA-sequencing was conducted to investigate the effects of sorgoleone, via expression of its biosynthesis pathway, on host gene expression. The production of sorgoleone in agroinfiltrated leaves as detected by gas chromatography/mass spectrometry (GC/MS) resulted in the formation of necrotic lesions, indicating that the compound caused severe phytotoxicity to these tissues. RNA-sequencing profiling revealed significant changes in gene expression in the leaf tissues expressing the pathway during the formation of sorgoleone-induced necrotic lesions. Transcriptome analysis suggested that the compound produced in vivo impaired the photosynthetic system as a result of downregulated gene expression for the photosynthesis apparatus and elevated expression of proteasomal genes which may play a major role in the phytotoxicity of sorgoleone.

Keywords: Nicotiana benthamiana; Sorghum bicolor; Sorgoleone; allelochemical; biosynthetic pathway; differential gene expression.

Fig. 1

Biosynthesis of sorgoleone. The enzymatic reactions starting from palmitoleoyl‐CoA are shown. Dihydrosorgoleone, the hydroquinone produced in vivo, undergoes auto‐oxidation once secreted into the soil to yield sorgoleone, a more stable benzoquinone. CYP71AM1, cytochrome P450; SbARS, alkylresorcinol synthase; SbDES, fatty acid desaturase; SbOMT, O‐methyltransferase.

Fig. 2

Physiological response to the transient expression of the sorgoleone biosynthetic pathway in Nicotiana benthamiana leaves. (a) A schematic diagram illustrates the details of the construct (pLH‐Sorg) containing five gene expression cassettes. (b) Necrotic lesions observed in leaf tissues infiltrated with Agrobacterium harboring the plasmid pLH‐Sorg at 5 d post‐infiltration (dpi) vs Agrobacterium bearing vector only as control (left). The younger leaves and stems above the infiltrated leaves were removed at the time the photos were taken. 35S‐Pro, 35S CaMV promoter; act‐Pro, Arabidopsis actin 2 promoter; act‐Ter, Arabidopsis actin 2 terminator; ARS2, S. bicolor alkylresorcinol synthases 2; CYP71AM1, S. bicolor cytochrome P450; DES2, Sorghum bicolor fatty acid desaturase 2; DES3, S. bicolor fatty acid desaturase 3; nos‐Pro, nopaline synthase promoter; nos‐Ter, nopaline synthase terminator; ocs‐Ter, octopine synthase terminator; OMT3, S. bicolor O‐methyltransferase 3.

Fig. 3

Gene expression cassettes for a set of constructs and the responses of Nicotiana benthamiana leaves to the expression of Sorghum bicolor genes. The binary vector pLH7000 was used to make all constructs for gene expression (see Supporting Information Methods S1 for details). (a) The cytochrome P450 CYP71AM1 expression cassette which was inserted into the binary vector to generate pLH‐G. (b) The expression cassettes for expressing desaturase 2 (SbDES2) and desaturase 3 (SbDES3) in the vector pLH‐DES. (c) The expression cassettes for the expression of alkylresorcinol synthase 2 (SbARS2) and O‐methyltransferase 3 (SbOMT3) in the vector pLH‐PQ. (d) The expression cassettes for expressing SbARS2, SbOMT3, and CYP71AM in the vector pLH‐GPQ. (e) The expression cassettes for expressing SbDES2, SbDES3, SbARS2 and SbOMT3 in the vector pLH‐PQD. (f–j) Nicotiana benthamiana leaves infiltrated with Agrobacterium harboring pLH‐G (f), pLH‐DES (g), pLH‐PQ (h), pLH‐GPQ (i), and pLH‐PQD (j), respectively. (k) Nicotiana benthamiana leaves infiltrated with Agrobacterium harboring pLH‐Sorg, which contains all five gene expression cassettes for sorgoleone biosynthesis. The leaves depicted in the panels on the left were infiltrated with Agrobacterium bearing the empty vector pLH7000 as control. Photos were taken at 5 d post‐infiltration (dpi).

Fig. 4

Development of leaf necrotic lesions. Leaves of Nicotiana benthamiana were infiltrated with Agrobacterium harboring pLH‐Sorg that contains five gene expression cassettes for sorgoleone biosynthesis (pictured in the images on the right) and the empty vector control (left). The development of the lesions was monitored daily: (a) 1 d post infiltration (dpi), (b) 2 dpi, (c) 3 dpi, (d) 4 dpi, (e) 5 dpi, and (f) 6 dpi.

Fig. 5

Sorgoleone content of agroinfiltrated Nicotiana benthamiana leaves. Sorgoleone concentrations were determined by gas chromatography/mass spectrometry (GC/MS) analysis of the extracts prepared from agroinfiltrated leaf areas expressing the sorgoleone pathway. Bars represent the means ± SD from assays performed in triplicate. nd, not detected.

Fig. 6

The global transcriptome response to the expression of the sorgoleone biosynthetic pathway in Nicotiana benthamiana leaves. (a) The number of differentially expressed genes (DEGs) at each time point. (b) Venn diagram showing the uncommon and the shared DEGs. (c) Gene Ontology (GO) annotation of differentially expressed genes. Classification of the DEGs was performed using the classification superviewer tool at the Bio‐Analytic Resource for Plant Biology (BAR; http://bar.utoronto.ca) with the default setting (P < 0.05). The classification of DEG sets of 3 through 5 d post infiltration (dpi) is shown.

Fig. 7

KEGG pathway classification of significantly enriched differentially expressed genes (DEGs). The statistical enrichment of differential expression genes in KEGG pathways was based on the results of analysis at g:profiler (Raudvere et al., 2019) with default settings and threshold P‐values set at 0.05. The data source for biological pathways was set to the KEGG database (KEGG FTP release 2020‐02‐03). The numbers of genes are indicated. dpi, days post infiltration.

Fig. 8

Major families of transcription factors (TFs) found in differentially expressed genes (DEGs). The area of the pie chart is scaled by total TF‐associated DEGs. ARF, auxin response; bHLH, basic helix‐loop‐helix; bZIP, basic leucine zipper domain proteins; C2H2, Cys2His2‐like zinc finger proteins; GATA/GRAS, GATA/GRAS domain family proteins; MADS, MADS‐domain proteins; MYB, myb domain proteins; NAC, NAM/ATAF/CUC domain proteins; WRKY, WRKY‐domain proteins.