Bacteriology Group, ICGEB Trieste

Intra- and Inter-cellular communication (quorum sensing) in plant-associated bacteria

In the laboratory of Dr. Venturi, the main topic of research is bacterial cell-cell communication of
plant-associated bacteria. This topic of research is very competitive, many laboratories around the
world are currently involved in studying how plant-pathogenic and plant-beneficial bacteria
communicate and synchronize their activities as this has evident potential practical applications.
Introduction
Many microorganisms produce small signaling molecules and sense their concentration in the
immediate close environment (Xavier, 2011). This signaling mechanism, called quorum sensing
(QS), relies on the accumulation of signal molecules within a population of bacteria and when it
reaches a threshold (quorum) the population responds in unison (Fuqua & Greenberg, 2002). The
population response is through the coordinated expression of specific target genes. A typical Gramnegative QS system involves the production of an acylated homoserine lactone signal (AHL) which
has been linked with regulating virulence factors, motility, plasmid transfer, nodulation, antibiotic
production and biofilm formation (Fuqua et al., 2001; Von Bodman et al., 2003; Whitehead et al.,
2001).
The rhizosphere (soil closest to plant roots) has been the focus of agricultural research for many
years due to its importance to crop productivity, soil health and sustainable agriculture
(Ordookhani et al. 2011). Rhizobacteria are microorganisms that colonize the rhizosphere at
different stages of plant development; some PGPR rhizobacteria are also able to colonize internal
tissues, these are called endophytes. Plant-Growth-Promoting Rhizobacteria (PGPR) and
endophytes produce beneficial effects on plant growth through several mechanisms such as
biological nitrogen fixation (Hurek et al. 2002), siderophore production (Hayat et al., 2010),
improved nutrient uptake (Kraiser et al. 2011), phytohormone production (Ashraf et al. 2011), and
induction of systemic resistance (ISR) (Bakker et al. 2007). A single bacterium may possess more
than one of these mechanisms (Ahmad et al. 2008). The use of PGPR and endophytes as
bioinoculants is now considered an important possible alternative to chemical fertilizers,
antibiotics, herbicides and pesticides (Tilak et al. 2005; Ordookhani et al. 2011).
Rhizospheric soil can be regarded as a reservoir of high diversity of QS-active microbes and AHLproduction of Gram-negative proteobacteria in particular. In addition, a rich pool of diverse AHLdegrading microbes also can be expected to be present in the rhizosphere (Effmert et al., 2012).
Thus, this natural hotspot of QS could be further exploited to achieve new possibly effective
biotechnological approaches to control crop pathogenic bacteria and to support crop growth.
Aims
Rice is the most important food crop and a staple diet for large proportion of world population.
Bacterial PGPR and endophytes constitute an important class of rice beneficial bacteria and studies
are only beginning to understand the composition, diversity and role of these bacteria in providing
growth and fitness advantage to rice. The main objective of this study is an extensive isolation,
identification and validation of endophytic properties of a few bacterial isolates obtained from
Italian rice varieties cultivated under submerged and dry conditions. The isolates are bacterial
endophytes from different tissues of rice plants at different stages of growth. Bacterial species
were identified using both culture-dependent and culture-independent methods and comparison
among these sets of results was carried out. The role if quorum sensing, plant-bacteria signaling
will be studied in these isolates. In addition possible interspecies interactions among the bacterial
endophytic isolates will be tested in order to assess the possibility of performing mixed inocula on
rice. It is believed that these strains are potential bioinoculants and now need to be subjected to
greenhouse and field trials
Approaches to be tested
1 Role of QS in bacterial endophytes

The cell-cell communication QS mediated by AHLs requires two proteins that belong to the LuxI
and LuxR protein families. LuxI-like proteins are AHL synthases, whereas LuxR-like proteins
function as sensors/regulators that form complexes with AHL and that in turn can affect gene
expression of QS-target genes (Decho et al., 2011). By using traditional genetics and state of the
OMICs technologies and its tools, the role of AHL QS in plant-bacteria interaction and the genes
regulated via AHL QS during the host colonization will be determined.
2 QS studies in interspecific interactions
If time allows, experiments will be performed on possible interspecies signaling and interactions
between the different bacterial rice endophytes. Interspecies interactions are now being more
intensively studied as it is accepted that bacteria live in close associate with plants as part of
complex multispecies communities. Studying stability and interactions between different species is
therefore becoming very important in order to understand how microbial consortia can colonize
and interact with plants.
Techniques and methods to be used
The proposed research will involve several methodologies of molecular biology including:
digestion with restriction enzymes, agarose gel electrophoresis, purification of DNA fragments,
ligation with T4 DNA ligase, end filling with Klenow fragment of DNA polymerase, Southern
hybridization, PCR and RT-PCR analysis and transformation of E. coli as described (Sambrook et
al., 1989). Analytical amounts of plasmids will be isolated as described (Birnboim, 1983), whereas
preparative amounts were purified with Qiagen columns. Total DNA from Gram-negative bacteria
will be isolated by the sarcosyl-pronase lysis method (Better et al., 1983).
Techniques of bacterial genetics including bi-parental and tri-parental conjugations, construction
of genomic knock-out mutants, transposon mutagenesis, transcriptional and translational fusions,
construction of gene banks as well as techniques of general microbiology including bacterial
growth in several growth media and analysis of several bacterial phenotypes are all techniques
regularly performed in the laboratory of Dr. Venturi.
OMICs techniques, which will be used, include genome sequencing (using Illumina platforms;
outsourced) and RNAseq analysis. Other specific techniques include purification and
characterization of AHLs by organic extraction, TLC, HPLC and mass spectrometry as described
(Steindler & Venturi, 2007).