Abstract
The stalk apoplast fluid of sugarcane contains different sugars, organic acids and amino acids that may supply the demand for carbohydrates by endophytic bacteria including diazotrophs P. tropica (syn. B. tropica) strain Ppe8, isolated from sugarcane, is part of the bacterial consortium recommended as inoculant to sugarcane. However, little information has been accumulated regarding this plant-bacterium interaction considering that it colonizes internal sugarcane tissues. Here, we made use of the RNA-Seq transcriptomic analysis to study the influence of sugarcane stalk apoplast fluid on Ppe8 gene expression. The bacterium was grown in JMV liquid medium (100 ml), divided equally and then supplemented with 50 ml of fresh JMV medium or 50 ml of apoplast fluid extracted from sugarcane variety RB867515. Total RNA was extracted 2 hours later, the rRNAs were depleted and mRNAs used to construct libraries to sequence the fragments using Ion Torrent technology. The mapping and statistical analysis were carried out with CLC Genomics Workbench software. The RNA-seq data was validated by RT-qPCR using the reference genes fliP1, paaF, and groL. The data analysis showed that 544 genes were repressed and 153 genes were induced in the presence of apoplast fluid. Genes that induce plant defense responses, genes related to chemotaxis and movements were repressed in the presence of apoplast fluid, indicating that strain Ppe8 recognizes the apoplast fluid as a plant component. The expression of genes involved in bacterial metabolism was regulated (up and down), suggesting that the metabolism of strain Ppe8 is modulated by the apoplast fluid. These results suggest that Ppe8 alters its gene expression pattern in the presence of apoplast fluid mainly in order to use compounds present in the fluid as well as to avoid the induction of plant defense mechanisms. This is a pioneer study showing the role played by the sugarcane apoplast fluid on the global modulation of genes in P. tropica strain Ppe8.
Highlights
The apoplast is defined as a highly dynamic compartment which connects roots to leaves in a continuum flow through the stems and it has been considered a very important channel related to the perception and transduction of signals from the environment to the symplast [1]
Considering the biotechnological importance of the bacteria to the sugarcane crop and the scarcity of information accumulated in relation to its interaction with plants, this study aims to explore the role of stalk sugarcane apoplast fluid on the gene expression of P. tropica strain Ppe8, expecting to identify bacterial genes regulated by the apoplast fluid compounds during in vitro interaction
The expression pattern observed by RNA-seq analysis for genes fliP1, paaF and groL in strain Ppe8 cultured in presence of apoplast fluid was confirmed by RT-qPCR (Table 3)
Summary
The apoplast is defined as a highly dynamic compartment which connects roots to leaves in a continuum flow through the stems and it has been considered a very important channel related to the perception and transduction of signals from the environment to the symplast (intracellular) [1]. It has been suggested that diazotrophs prefer the plant apoplast fluid since the symplast restricts ions, sugar and other solutes due mainly to the permeability of the membrane [2]. Isolates of Azospirillum have been found within surface disinfected roots of sugarcane, suggesting that it endophytically colonizes sugarcane [5]. It has been suggested that apoplast fluid is one of the most suitable niches for baterial endophytes [7, 8]. In addition to reducing the competition, the apoplast fluid can supply various organic and inorganic compounds
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