Genome Mining and Gene Expression Reveal Maytansine Biosynthetic Genes from Endophytic Communities Living inside Gymnosporia heterophylla (Eckl. and Zeyh.) Loes. and the Relationship with the Plant Biosynthetic Gene, Friedelin Synthase

Abstract

Even though maytansine was first discovered from Celastraceae plants, it was later proven to be an endophytic bacterial metabolite. However, a pure bacterial culture cannot synthesize maytansine. Therefore, an exclusive interaction between plant and endophytes is required for maytansine production. Unfortunately, our understanding of plant-endophyte interaction is minimal, and critical questions remain. For example: how do endophytes synthesize maytansine inside their plant host, and what is the impact of maytansine production in plant secondary metabolites? Our study aimed to address these questions. We selected Gymnosporia heterophylla as our model and used amino-hydroxybenzoic acid (AHBA) synthase and halogenase genes as biomarkers, as these two genes respond to biosynthesize maytansine. As a result, we found a consortium of seven endophytes involved in maytansine production in G. heterophylla, based on genome mining and gene expression experiments. Subsequently, we evaluated the friedelin synthase (FRS) gene's expression level in response to biosynthesized 20-hydroxymaytenin in the plant. We found that the FRS expression level was elevated and linked with the expression of the maytansine biosynthetic genes. Thus, we achieved our goals and provided new evidence on endophyte-endophyte and plant-endophyte interactions, focusing on maytansine production and its impact on plant metabolite biosynthesis in G. heterophylla.

Keywords: AHBA synthase gene; FRS gene; endophyte–endophyte interaction; halogenase gene; maytansine-producible endophytes; plant–endophyte interaction.

Figure 1

Chemical structure of maytansine (left) and 20-hydroxymaytenin (right).

Figure 2

Experimental scheme showing the design and techniques applied in this study.

Figure 3

PCR products of the amplified AHBA synthase gene, spanning around 755 bp in different parts of 2017 and 2018 collections of G. heterophylla, originating from South Africa, compared to A. mirum (DSM 43827) as the reference. Our reference, A. mirum (AM), is indicated by 0, while 1 indicates the aerial part of G. heterophylla cultivated at our laboratory in Germany (GH-Lab). Furthermore, 2 to 4 indicate the leaves, stems, and roots of the 2017 collection of G. heterophylla, originating from South Africa (2017-SeL, 2017-SeS, and 2017-SeR), while 5 to 7 indicate the counterpart from the 2018 collection (2018-SeL, 2018-SeS, and 2018-SeR).

Figure 4

PCR products of the amplified halogenase genes, spanning around 550 bp in different parts of 2017 and 2018 collections of G. heterophylla originated from South Africa, compared to A. mirum (DSM 43827) as the reference. Of note: 0 indicates A. mirum (AM) while 1 indicates the aerial part of G. heterophylla cultivated at our laboratory in Germany (GH-Lab). Additionally, 2 to 4 indicate the leaves, stems, and roots parts of the 2017 collection of G. heterophylla originated from South Africa (2017-SeL, 2017-SeS, and 2017-SeR), while 5 to 7 indicate counterparts from the 2018 collection (2018-SeL, 2018-SeS, and 2018-SeR). The left-handed figure indicates the 1st amplification, while the right-handed one indicates the 2nd amplification, both from the isolated 1st PCR products (red boxes).

Figure 5

Phylogenetic tree of the obtained halogenase gene sequences from G. heterophylla, and homologous sequences from the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 15 October 2021). This tree is deposited on the TreeBASE database (TreeBASE ID: Tr134221). Multiple alignments are performed using the Muscle tool, and the phylogenetic tree is generated using the UPGMA model from MEGA-X software (Version 10.0.4). The bootstrap values are shown on the branch based on 1000 pseudoreplicates. The distances of this evolutionary tree are calculated using the Maximum Composite Likelihood method. The halogenase gene from Sinomenium acutum is used as a plant gene.

Figure 6

Maytansine’s biosynthetic genes expression. AHBA synthase gene expression is presented on the left (A), while halogenase gene expression is shown on the right (B). The relative gene expression is exhibited in the mean ± SD of triplicated samples (n = 3). AM indicates A. mirum (DSM 43827) as a reference. GH-Lab indicated G. heterophylla cultivated in our laboratory, Germany. 2017-SeL, 2017-SeS, and 2017-SeR indicate the leaves, stems, and roots parts of the 2017 collection of G. heterophylla originated from South Africa, while 2018-SeL, 2018-SeS, and 2018-SeR indicate the counterpart from the 2018 collection. Additionally, 16S rRNA is used as a housekeeping gene. The bar graph uses the x-axis on the right side while O indicates each sample data point, using the x-axis of the left side. * indicates the statistic significant by student t-test (one-tail) with p-value ≤ 0.05. ** indicates the statistic significant by one-way ANOVA with p-value ≤ 0.05. ^¥ indicates the highest AHBA synthase gene expression.

Figure 7

FRS gene expression of G. heterophylla from our laboratory and the 2017 and 2018 collections originating from South Africa. GH-Lab indicates G. heterophylla cultivated in our laboratory, Germany; 2017-SeL, 2017-SeS, and 2017-SeR indicate the leaves, stems, and roots of the 2017 collection of G. heterophylla originating from South Africa; 2018-SeL, 2018-SeS, and 2018-SeR indicate the counterparts from the 2018 collection. Additionally, 40S ribosomal protein is used as a housekeeping gene. The bar graph uses the x-axis on the right side while O indicates each sample data point, using the x-axis of the left side. ** indicates the statistic significantly by one-way ANOVA with p-value ≤ 0.05. ^¥ indicates the highest FRS gene expression level.

Figure 8

A six-month-old vegetatively cloned G. heterophylla plant from a stem of G. heterophylla collected in 2018, originating from South Africa (left). PCR products of the amplified AHBA synthase genes spanning around 755 bp in the different parts of the cloned G. heterophylla (middle). PCR products of the amplified halogenase genes spanning around 550 bp in the different parts of the cloned G. heterophylla (right). AM indicates A. mirum (DSM 43827) as the reference. Cloned-SeL, cloned-SeS, and cloned-SeR indicate leaves, stems, and roots of the vegetatively cloned G. heterophylla.

Figure 9

Proposed mechanistic response and relationship between the maytansine biosynthesis from endophytic communities and the 20-hydroxymaytenin biosynthesis from G. heterophylla, a plant host. A indicates the downregulation of the AHBA synthase gene and the activation of the halogenase from endophytes in different organs of G. heterophylla, and B indicates the upregulation of the FRS gene from the 20-hydroxymaytenin biosynthesis in the G. heterophylla roots. Blue arrows indicate the flow of AHBA transportation from the leaves to roots.