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Transgenic poplars: Is horizontal gene transfer from Agrobacteria to endophytic bacteria possible?

(2005 – 2008) Federal Research Centre for Forestry and Forest Products (BFH), Institute for Forest Genetics and Forest Tree Breeding, Waldsieversdorf

Topic

The poplar was used as a model to investigate the possibility of gene transfer from recombinant Agrobacteria to endophytic bacteria that occur in poplars. These bacteria colonise plant tissue, including that of woody forest plants. Not much is known about the incidence of endophytic bacteria. During plant transformationand afterwards, the fact that the recombinant Agrobacteria are in the immediate vicinity of the endophytes means that recombinant DNAis transferred to the endophytic bacteria.

Since woody forest plants live a long time, the probability of horizontal gene transfer (HGT) is increased.

The project had a focus on the following areas

  • Isolation and characterisation of endophytic bacteria from various poplar clones
  • HGT in a model: In-vitro research into horizontal gene transfer from Agrobacteria to endophytic bacteria
  • HGT detection in the poplar: Identifying and quantifying endophytic bacteria that carry the transgene.
  • Research into the persistence of the Agrobacteria in transgenic poplars and development of methods to reduce the number of endophytes.

Summary

Culture-dependent and culture-independent methods were used to characterise the endophyte community. Field poplar clones and in-vitro propagation systems were analysed.

  • The endophyte community in the field poplar clones was found to be generally very diverse.
  • Significant differences were found in the composition of the endophyte community between poplar clones and depending on the location.
  • Conjugation between bacteria is the most common event leading to horizontal gene transfer. When this occurs, plasmid DNA is transferred from a donor organism to a recipient via a ‘cell bridge’. Horizontal gene transfer was detected only when conjugation was triggered by an external plasmid.
  • In the in-vitro propagation systems the main bacteria found were isolates of the genus Paenibacillus. Since Paenibacillus is far removed from Agrobacterium in phylogenetic terms and therefore does not have any plasmids that could trigger conjugation, gene transfer from Agrobacterium during or after transformation can practically be ruled out.
  • Persistent Agrobacteria were detected up to three months after transformation in vitro, and 16 months after transformation in one poplar line.
  • The probability of horizontal gene transfer falls when there are fewer endophytic bacteria on the plant being transformed. Regeneration from shoot and root meristems removed under sterile conditions is an effective method of obtaining plants free from endophytes.

Experiment description

Leaf pieces are infected with Agrobacteria

small pieces of leaves, infected with Agrobacteria

Regeneration of buds that form at the wound site

regeneration of buds, that are formed at the sore spot

Endophytic bacteria that live in the plant tissue. In order to insert new genes into plant cells the soil bacterium Agrobacterium tumefaciens is used as a “gene ferry”. If genetically modified Agrobacteria survive in the plant, gene transfer co

endophytic bacteria

Photograph: Dietrich Ewald

Material

Poplars: A female poplar hybrid of Chinese origin was used for the transformation. Clones of this hybrid have no reproductive ability, so the hybrids cannot be spread via seeds.

Agrobakterien: For the transformation of the poplar clones, an Agrobacterium tumefaciens strain was used with a binary vector that carries a GUS reporter gene. This made it possible to check whether the transfer of the binary vector had taken place.

Isolation and characterisation of endophytic bacteria

The endophytes were isolated from poplar leaf and shoot parts. The surface of these had to be disinfected to eliminate any colonizing bacteria. The plant material was then homogenized, plated at various degrees of dilution onto culture media and cultivated until the endophytes grow.

The bacterial isolates were identified using PCR (amplification and sequencing of the 16S rDNA). Morphological and physiological tests were also carried out.

Culture-independent analysis of the endophytes

Not all endophytic bacteria of the poplar can be cultivated. By amplifying certain bacterial DNA segments (16S rDNA) straight from the plant material and setting up a clone library, it is possible to record these bacteria as well. Characterisation then took the form of sequencing the 16S rDNA.

Research into horizontal gene transfer between Agrobacteria and endophytes

Conjugation between bacteria is the most common event that leads to horizontal gene transfer. A ‘cell bridge’ (conjugation) leads to the specific transfer of plasmid DNA from a donor to a recipient organism.

Model system for HGT: The possibility of a horizontal gene transfer (HGT) to endophytic bacteria occurring during plant transformation with binary vectors was to be investigated using a model system. Among other issues the study with test whether a transfer can result through the activation of the co-called virulence gene (vir-gene), which is a part of the binary vector.

Identifying HGT in transformed poplars: Leaf and shoot material from transgenic poplars was tested for the presence of endophytes that have received the transgene from the Agrobacteria through conjugation. Samples were taken at different times: immediately after transformation of the poplars, after three and six months and after one and seven years. The isolated endophytes were tested for the presence of the binary vector (transconjugants).

Persistence of Agrobacteria

It is assumed that the probability of HGT increases if the Agrobacteria used for the transformation persist for a long time in the plant. For this reason, the plants were tested for the presence of persisting Agrobacteria using classic detection methods (isolation and cultivation) and PCR (detection of non-cultivable bacteria), in a similar way to the endophyte testing at different times. In order to further reduce the possibility of HGT, a method was developed that reduces the number of culturable endophytes in vitro.

Results

Isolation and characterisation of endophytic bacteria from various poplar clones

The endophyte community in the field poplar clones was found to be very diverse. As many as fifty different genera of Actinobacteria, Proteobacteria, Firmicutis and Bacteroidetes were found. There was a clear difference between the classes of bacteria found using the culture method and those found using the culture-independent method. Whereas most of the culturable bacteria found were Actinobacteria, the dominant bacteria in the culture-independent method were Proteobacteria.

Starting with a greenhouse poplar, it is possible to culture a shoot meristem.

Shoot meristem: cultured tissue from a shoot

Root meristem

Root meristems can be cultured from e.g. shoots with roots

Root meristem: In order to reduce the number of endophytic bacteria, plants are regenerated from meristems. Meristem tissue consists of undifferentiated plant cells that are capable of dividing infinitely, such as those found at the growing tip of

Significant differences in the distribution of the bacteria groups were found not only between the two methods, but also between the individual poplar clones. The location of the clones also had an influence on endophyte diversity.

In contrast with the high diversity in the outdoor poplar clones, in the in vitro propagation systems the most common isolates found were of the genus Paenibacillus. Since Paenibacillus and Agrobacterium are far removed from each other in phylogenetic terms, the probability of a gene transfer from Agrobacterium is very low.

Reduction in number of endophytic bacteria

Experiences with in vitro cultivation in fruit farming show that meristems isolated from woody fruit plants have a lower density of viruses. In conjunction with the team’s own research (results of the in vitro cultivation), the meristem culture (shoot and root meristems) method was therefore chosen to reduce the number of endophytes. Sampling was carried out three, seven, eleven and 19 months after culturing the meristem from a shoot and the subsequent regeneration of plants in vitro. No endophytes were found at any time using any of the methods.

HGT detection

Horizontal gene transfer of binary plasmids from recombinant Agrobacteria to endophytic bacteria (Stenotrophomonas, Agrobacterium rubi) has so far been detected only in vitro. The binary vector was transferred to endophytic bacteria (Stenotrophomonas, Agrobacterium rubi) using an external plasmid. By contrast, no vir-gene-induced transfers have been detected so far.

Persistence of Agrobacteria

Agrobacteria were detected in the poplar lines up to three months (in one case 16 months) after transformation. Once the transformed plants had been moved to the greenhouse, Agrobacteria were found again in several samples.