Impact of glyphosate application to transgenic Roundup Ready soybean on horizontal gene transfer of the EPSPS gene to Bradyrhizobium japonicum and on the root-associated bacterial community

Abstract In this study, two topics causing major public concern
related to transgenic plants were investigated: The possibility of
a horizontal gene transfer from plant to bacteria and the impact of
transgenic plants after herbicide treatment on root associated
bacteria. The transgenic plant chosen for this study was Roundup
Ready (RR) soybean, which is tolerant to the herbicide glyphosate
and is the most commonly used genetically modified crop worldwide.
Glyphosate, the active ingredient of Roundup Ready, inhibits the
EPSPS enzyme (5-enolpyruvylshikimate-3-phosphate synthase). EPSPS
is an enzyme involved in the shikimic acid pathway leading to the
aromatic amino acid biosynthesis and its inhibition leads to growth
reduction of plants and microorganisms. RR crops are glyphosate
tolerant due to the introduction of the CP4-EPSPS gene coding for a
glyphosate insensitive EPSPS enzyme. The transgenic construct is
under expression of a CaMV 35S promoter a nos transcriptional
termination element from Agrobacterium tumefaciens. Horizontal gene
transfer experiments with the EPSPS gene of the RR soybean were
performed under controlled laboratory conditions and were targeted
to the nitrogen fixing symbiont of soybean Bradyrhizobium
japonicum. This bacterium comprises the requirements of a possible
receptor for the glyphosate resistance trait, as it is sensitive to
the herbicide and thus the acquirement of glyphosate resistance
would signify a positive adaptation to glyphosate accumulated in
the roots after herbicide application. Two key conditions for gene
transfer from the CP4-EPSPS gene from the RR soybean to B.
japonicum were evaluated in this study: The required specific
conditions for B. japonicum to undergo natural transformation and
the expression of the CP4-EPSPS gene in B. japonicum. For that
purpose, the CP4-EPSPS gene was cloned into a B. japonicum
chromosomal integration vector and was transferred by biparental
mating into the B. japonicum genome. Subsequently, the expression
of the CP4-EPSPS gene in B. japonicum was tested under increasing
glyphosate selection pressure. Results of these experiments
indicated that B. japonicum is not naturally transformable under
any conditions known from the more than 40 so far reported
naturally transformable bacteria. Furthermore, the CP4-EPSPS
genetic construct, as contained in RR soybean, has been shown in
this study to be not active in B. japonicum. Consequently, if there
would be a gene transfer of the plant CP4-EPSPS to B. japonicum,
this genetic construct does not confer glyphosate resistance to B.
japonicum and does not constitute any adaptive advantage to the
bacterium under glyphosate selection pressure. As the genetic trait
of glyphosate resistance has been found in several bacteria, it
would be more probable that the common mating exchange between
bacterial groups could disperse the glyphosate resistance within an
environment. Moreover, in the specific case of B. japonicum, a high
spontaneous mutation rate for glyphosate resistance was observed,
suggesting that B. japonicum can also adapt to the glyphosate
selection pressure by mutation under natural conditions. The impact
of transgenic plants with their respective herbicide treatments on
root associated bacteria was investigated in a greenhouse
experiment. The composition and diversity of bacterial communities
of RR soybean rhizospheres were analyzed and compared between
glyphosate-treated and untreated plants. Samples from five harvests
with two glyphosate applications were analysed by 16S rRNA gene
T-RFLP analysis complemented with the evaluation of three clone
libraries. Multivariate statistical analysis of the data was used
to visualize changes in the microbial populations in response to
glyphosate applications and in order to find groups of organisms
responsible for the observed community shifts. A comparison of the
rhizosphere communities revealed that a Burkholderia related group
was significantly inhibited by glyphosate application, while the
abundance of a group of Gemmatimonadetes related sequences
increased significantly after the herbicide treatment. The
significant increment of Gemmatimonadetes abundance after
glyphosate application could indicate that these organisms are able
to metabolize the herbicide. Shannon diversity indices were
calculated based on the T-RFLP results with the aim to compare
bacterial diversity in the rhizosphere of glyphosate-treated and
non treated RR soybeans. Interestingly, the bacterial community
associated to RR soybean roots after glyphosate application not
only demonstrated effective resilience after the disturbance but in
addition the bacterial diversity also increased in comparison to
the untreated control samples. It is possible, that in an
environment with organisms which are able to metabolize glyphosate,
the key for enhancing diversity could be the succession of
metabolites, which can be further utilized by a diverse range of
bacteria.