ECF sigma factors: How endophytes sense the plant

Endophytes are fungi or bacteria that spend at least one phase of their life span inside a plant without causing disease symptoms to their host. Most endophytes originate from the rhizosphere microflora. During transition from the soil to the plant environment bacteria may face rapidly changing conditions, causing most probably severe stress. The bacterias survival may depend very much on its ability to adapt to the new environment. An in-time adaptation might be achieved by switching the genetic programs in response to the external signals. Within this project we aim at answering the question whether bacterial endophytes use cell surface signaling mechanisms, based on extracytoplasmatic function (ECF) sigma factors, to sense stimuli from the host plant and to adjust their gene expression. As model organism we will use the plant-growth promoting endophyte and rhizobacteria B. phytofirmans PsJN. We plan to isolate and characterize the genes coding for ECF sigma factors in strain PsJN. Quantitative PCR amplification of ECF sigma factor genes will be used to identify ECF sigma factors that respond to plant signals. This ECF genes will be disrupted and mutants tested for their ability to establish populations in and to promote growth of micropropagated potato plantlets. Overexpression of ECF genes and subsequent transcriptome comparison to a not induced control will identify genes that are regulated by a given ECF sigma factor in response to plant stimuli. Finally, results will be verified by in vivo expression analysis of genes that are in control of given ECF sigma factors.

Endophytes are fungi or bacteria that spend at least one phase of their life span inside a plant without causing disease symptoms in their host. Today, it is widely accepted that endophytes actively colonize plants and interact with its host and positive effects on plants growth and stress resistance are well documented. However the mechanisms of plant-bacteria communication are still poorly understood. How for example do bacteria recognize the plant environment? Do they feel plant stress and how do bacteria respond to plant stimuli? One way how bacteria sense and react to the extracellular environment is the so-called cell surface signalling (CSS) employing alternative sigma factors. This signal transduction system consists of a receptor in the outer cell membrane, an inner-membrane bound regulator and bound to that an extracytoplasmatic function (ECF) group IV sigma factor. Upon signal recognition the ECF sigma factor is released and activates expression of its target genes. Within this project we aimed at answering the question whether bacterial endophytes use cell surface signaling mechanisms, based on extracytoplasmatic function (ECF) sigma factors, to sense signals from the host plant and to adjust their gene expression and how bacterial endophytes respond to plant stress. We conducted experiments that allowed for identification of ECF sigma factors in B. phytofirmans PsJN that are activated in response to plant stress. Six ECF sigma factor genes were expressed in PsJN colonizing plants and the expression of one ECF sigma factor was significantly up-regulated when the plant was stressed. This indicates that B. phytofirmans PsJN actively senses the conditions in the host plant and therefore uses cell-surface signaling systems employing alternative sigma factors. We analyzed also the genetic response of B. phytofirmans PsJN to plant stress by whole- transcriptome sequencing. Transcripts significantly up-regulated in response to induction of plant drought stress were mainly involved in transcriptional regulation, cellular homeostasis and the detoxification of reactive oxygen species whereas genes of the general metabolism were down-regulated. These data suggest that B. phytofirmans PsJN tried to protect it-self from oxidative stress. This may be explained by the fact that abiotic stress provokes immune defense reactions in plants such as the production of high amounts of reactive oxygen species. In this context, our study indicates that the endophyte B. phytofirmans PsJN is affected by plant drought stress, that it senses plant stress signals and adjusts its gene expression accordingly.