Neha Sharma Punjab Agricultural University

1. REPORTER GENES FOR THE STUDY
OF RHIZOBIAL ECOLOGY
Presented by-
Neha Sharma
Punjab Agricultural University

2. INTRODUCTION
 Some beneficial genera Rhizobium, Bradyrhizobium and
Azorhizobium, forms nodule and fixes atmospheric nitrogen
 A sequence of events allows the establishment of a
symbiotic state where nitrogen is fixed and assimilated into
organic nitrogenous compounds
 Strains generally faced various abiotic and biotic stresses
 The phenomenon of strain competition is poorly understood
 The ecology of rhizobia in the soil is crucial to the
establishment and performance of the legume-rhizobium
symbiosis

3. ABIOTIC FACTORS
 Temperature-
The elevated temperatures delay nodule initiation, nodule development,
and interfere with nodule functioning
 Moisture:
Very low or very high soil moisture contents are harmful for rhizobia
 Soil pH:
Extremes of pH frequently reduce the nodule formation by reducing the
colonization of soil and the legume rhizosphere by rhizobia
 Soil salinity:
High external salts induce an increase in the intracellular pool of amino
acids helps in survival
 Available nutrients:
Correlation between the soil organic matter levels and the size or strain
composition of populations of rhizobia have been reported

4. BIOTIC FACTORS
 Competition:
The ideal rhizobial strain for inoculation should have the ability
to persist and grow in the soil or around plant roots. The highly
effective strains of rhizobia generally fail to grow under natural
soil condition may be due to inter-strain competition
 Antagonism:
Many strains of rhizobia produce bacteriocins which inhibit the
growth of the related strains
 Predation and parasitism:
Bdellovibrio bacteriovorus parasitizes certain rhizobia and
cause their lysis.
Exposure of sensitive rhizobia to phages leads to selection of
variants with reduced sensitivity to phage lysis.

5. APPROACHES TO STUDY THE ECOLOGICAL
FACTORS
 Indirect methods-
i. By accessing the proportion of nodules by ELISA,
DNA hybridization.
ii. Identifying rhizobia by antibiotic resistance,
bacteriophage typing, PAGE, immunoblot
techniques.
 Direct quantitative measurements-
Plant infection method, fluorescent antibody
technique, antibiotic marker technique, genetic
markers and non-radioactive DNA hybridization

6. REPORTER GENE
 A gene which is attached to a regulatory sequence
of another gene of interest in bacteria, cell culture,
animals or plants
 used to study the role of a microorganism in
ecosystem
 to investigate the expression of other genes for
which functional assays are not available or are
difficult.

8. REGULATION OF REPORTER GENE

9. CONSTRUCTION OF MINI-TRANSPOSONS
 Tn5 transposable element is used.
 Herrero et al. (1990) and de Lorenzo et al. (1990)
constructed mini-transposons, for the insertion of reporter
gene and transferred from E. coli to Rhizobium through
conjugation.
 An additional transposase gene was attached external to
the transposon to increases stability and acceptability in
Rhizobium .

10.  Gene cassette- consists of the marker gene itself and
of sequences that regulate gene expression. Such
sequences are primarily known as promoters and
terminators that are essential for switching on and off
gene expression.
 Promoters may be regulated, generally either by gene
products of other regulating sequences or environmental
signals.

11. 1. gusA
 isolated from E.coli
 gene product- glucuronidase
 Substrate- X-glcA (5-bromo-4-chloro-3-indolyl-glucuronide),
Magenta-glcA (5-bromo-6-chloro-3-indolyl-β-D-glucuronide)
 gusA-marked cells turn blue when the washed root is incubated in a
phosphate buffer containing a GUS substrate such as X-glcA
 Useful for the study of Rhizobium, Bradyrhizobium, Agrobacterium,
Azospirillum, Pseudomonas, Streptomyces competition and plant
colonization
 Advantage-
i. Easy to perform
ii. eliminates the time-consuming steps of picking nodules and
preparing bacterial isolates
iii. Can be detected by the visualization method

12. mTn5SSgusA10-
 tac promoter regulate the expression, with the
repressor gene.
 Advantage- marker gene is not induced without
inducer IPTG (isopropyl-ß-D-thiogalactoside)
mTn5SSgusA11-
 the marker gene is constitutively expressed
 It is used to detect rhizobial strain in soil and
rhizosphere

13. mTn5SS gusA31
 used for symbiotic expression study of nodule
occupancy
 promoters of the nifH gene codes for the Fe-
component of the enzyme nitrogenase
 Gene expression occurs in symbiotic conditions
mTn5SSgusA20-
 aph promoter- kanamycin resistance gene,
function in Gram -ve bacteria
 constitutive gusA expression
 Used for rhizosphere colonization

14. IN BACTERIA-LEGUME SYMBIOSIS
• gusA gene from Escherichia coli with plasmid
Tn5gusA1 & Tn5gusA2 to study the expression of
Rhizobium meliloti symbiotic genes (nod,nif,fix)
during the nodule organogenesis of alfalfa was used.
• To detect the expressed gene activity histochemical
assay of β-glucronidase was done

15.  The nod genes, involved in nodule formation
expressed in the early phase (after 36 hour of
inoculation) on root surface
 The nif ,fix genes were expressed 10 days after
inoculation in the active symbiotic zone of nodules.

16.  Similar study in pigeon pea roots-
inoculation of pigeon pea root by a marked strain
Rhizobium sp.234 :gusA31,
unmarked sp. of Rhizobium sp.234:gusA11
Result- Marked strains of gusA31 showed activity
in the central region (nitrogen fixing zone) of the
nodule.
Rhizobium sp. marked with mTn5SSgusA11
showed gus activity to the outer region of the
nodule where new infection take place.

17. NODULE OCCUPANCY COMPETITION ASSAY
Pigeon pea plant inoculated with-
Rhizobium sp.NGR234 (unmarked)
Rhizobium sp. NGR234 :gusA31(marked)- 7:1.
Again, 1:15.
Result- The proportion of blue nodules increased with an
increased proportion of the gus marked strain.

18. gus reporter gene in the study of PGPR in
rhizosphere
 To study the Azospirillum on wheat roots in colonization
assays pFAJ31.13-
 Inserting A. brasilense gene upstream of a promoter
less reporter gene.
 Result-
i. After inoculation, the bacterial cells were found in the
root hair zones and sites of lateral root emergence.
ii. A. brasilense mutants , impaired in motility,
chemotaxis and plant root attachment
It was concluded that some key compound are secreted
by the root hair zone of the wheat roots to which
Azospirillum is attracted and colonized.

19. 2. lac Z reporter gene
 Codes for ß-galactosidase enzyme involved in the
metabolism of lactose in to glucose and galactose.
 Colonies with lacZ gene can be identified on X-Gal
plates gives blue colour and can also be identified
on ONPG (ortho-nitrophenyl-β-galactoside) and
MUG (4 methylumbelliferyl-β-D-glucuronide)
substrates, which can be measured by yellow color
spectrophotometrically.
 It is used to study rhizobial competition, root
colonization, gene expression, survival.

20. LAC Z FUSIONS
promoterless lacZ
beta-galactosidase
substrate colour change
mRNA
promoter from test gene

21. XYLE REPORTER GENE
• Xyl E gene encodes for 2,3catechol dioxygenase .
• Convert catechol ( colorless ) to 2- hydroxymuconic semi
aldehyde, a bright yellow pigment that can be visualized
or measure spectrophotomitrically.
• The gene is originally found on TOL plasmid in P. putida.
• Colony grown on media sprayed with catechol, positive
clones turn bright yellow.
• Product is soluble.
• The xylE gene used to study in situ gene expression .

22. LUXAB(BIOLUMINESCENCE)
 luxAB gene code for the enzyme luciferase.
 Bacterial luciferase catalyze a light emitting oxidation
reaction using O2 ,FMNH₂ and a long chain aliphatic
aldehyde (decanel).
 Both o₂ and FMNH₂ are present in most bacterial cell so any
bacterium can produce light if contain lux AB gene if decanel
provide exogenously.
 Photinus pyralis(firefly) have the gene luc that also use as a
reporter gene.

23. LIGHT EMISSION IS DETERMINED BY A LUMINOMETER.

24. CEL B GENE
• Encode for thermostable β-galactosidase activity.
• It allow detection of the marker strain after heat-
inactivation of the endogenous enzyme.
• Isolated from thermophilic archeaon Pyrococcus furiosus.
• Half life 85 h at 100 0C
• Assay for detection:
• The washed roots are incubated in phosphate buffer at
700C in order to destroy endogenous enzymes.
• Roots are incubated in the presence of a chromogenic
substrate such as X-gal. Nodules containing the celB
marked strain turn blue

25. GREEN FLUORESCENT PROTEIN(GPF)
 GFP is isolated from Pacific jellyfish Aequorea victoria,
and Sea pansy Renilla reniformis.
 GFP is a protein composed of 238 amino acid residues
(26.9 kDa) that exhibits bright green fluorescence when
exposed to light in the blue to ultraviolet range.
 GFP protein is coded by gfp reporter gene.

26. 1

27. USE OF GFP TO STUDY THE TRICHODERMA-PATHOGEN-
PLANT INTERACTION
 Trichoderma atroviridae strains are use as biocontrol
(biofungicide) agent for Rhizoctonia solani.
 The antagonistic activity is attributed by nutrient competition,
antibiosis, the activity of cell wall lytic enzymes.
 To study the antagonistic activity of Trichoderma we use gfp
activity.
 For this experiment they use:
 gfp tagged mutants of T. atroviride.

28. MYCOPARASITISM BY T.ATROVIRIDE IN COCULTURE
(A)T. atroviride hyphae grew towards the host hyphae.
(B)T. atroviride hyphae aligned with R. solani hyphae.
(C) R.solani broken hyphae during attack by T.
atroviride. This mode of attack was not observed with
the other fungal host.

29. (d) T. atroviride hyphae grew alongside with hyphae &
branched towards adjacent hyphae.
(e) T. atroviride spores adhered to the host hyphae &
germinated after attachment.
(f) The young T. atroviride hyphae parasitized the host
mycelium & also the T. atroviride hyphal tips swelled and form
papilla like structures.

30. THANK YOU