1. The Pests Under Our Feet and What They Do To What We Eat
Ethan Barach, Michelle Perry, Mentor: Dr. Heather Marella,
Department of Biological Sciences at Bridgewater State University
IntroductionMeloidogyne incognita is a parasitic nematode that is capable of infecting thousands of plant species.
It is more commonly known as the “cotton rootknot nematode”. It is an obligate endoparasite. This
means that it needs to exploit a host in order to complete its life cycle and that it infects from inside
the host. Meloidogyne species are responsible for billions of dollars of crop damage worldwide. They
can be found in temperate, tropical, and sub-tropical regions of the world. The root-knot nematode
reproduces asexually but during times of stress, such as a drought, the nematode will produce males
that leave the plant host and die shortly after.
Arabidopsis thaliana is a relative of crop species such as cabbage and radishes. It is native to Asia,
Europe and Northwest Africa. Arabidopsis was used for this experiment because its genome is
relatively small and easy to work with, it has simple cell biology, a well understood transformation
system, and there are lots of molecular resources online regarding this plant. Mutant Arabidopsis
seeds were received from the Arabidopsis Biological Resource Center with mutations in ATP binding
cassette (ABC) transporters. ABC transporters serve a primary function of exuding phytochemicals
out of the roots into the soil. These chemicals in a normal plant can serve as an attractant to parasitic
nematode species that follow the chemical gradients given off by the roots. More specifically
mutations in the AAP6 and AAP3 genes (amino acid transporters) have been shown to have
significant reduction of parasitic nematode infections in previous research (Marella, 2012).
The goal of this experiment was to observe the effect of the mutant ABC transporter gene At4g15320
(Atpdr2 ) had on the root-knot nematode compared to the Columbia wild type Arabidopsis. The
success of the parasitism was determined by the number of female nematodes found in roots of the
plants and infectivity by stage 2 juveniles. Adult male nematodes were also counted because this
would signify that the mutation is causing stress for the nematode. The mutation of the ABC
transporter was also tested by PCR to determine if the mutation was homozygous or heterozygous in
the plant and whether sufficient transcript was being made to make a functional protein.
AbstractMeloidogyne incognita is an endoparasitic nematode that infects thousands of plant species worldwide and causes billions of dollars in crop damage. Due to this agricultural problem, research is conducted to identify plant strains that will be resistant to infection. Arabidopsis thaliana is the plant species being used in our research due to its simple
transformation system, small genome, and the large amount of molecular tools available. Arabidopsis seeds mutated in gene At4g15230 were obtained from the Syngenta Arabidobpsis Insertion Library (SAIL). The gene encodes an ABC transmembrane transporter involved in the movement of phytochemicals. A mutation within this locus can effect the
expression of that gene thereby interfering with a vital process within the plant transport system. Seeds were sterilized and sown on to Phytagel plates for seedling growth. Some of the Arabidopsis plates were infected with nematode eggs for counting adult males and females after 5-7 weeks of infection. Other plates of seedlings were used to isolate nucleic
acids. DNA was extracted from the seedlings and T-DNA verification was performed using PCR to confirm the mutation. Meloidogyne incognita stage 2 juveniles were added to the Arabidopsis and allowed to infect for 1 day or 7 days. Acid fuchsin staining was then performed to assess the number of nematodes infecting the roots. RNA from the mutant
was extracted to make cDNA. The cDNA was used for PCR to determine the expression level of our gene. Finally, we counted the number of adult male and female nematodes produced on the mutant plants
Materials and Methods
•Seed Sterilization: Atpdr2 mutant and Columbia WT Arabidopsis thaliana seeds were sterilized
with 70% ethanol, and plated on Phytagel plates. Seedlings grew for 6-8 weeks and some were
infected with nematode eggs and others were used for nucleic acid isolation.
• DNA Extraction: DNA from Atpdr2 mutant and Col WT seedlings were extracted with Qiagen
DNeasy plant mini kit protocol and performed using liquid nitrogen.
• DNAAmplification: A Polymerase Chain Reaction (PCR) protocol from New England Biolabs’
Quick-load Taq 2X Master mix was followed for DNA amplification. Thirty cycles were performed
at 51C annealing temperatures. Both DNA types were tested with primers for the intact genes and
examined for T-DNA insertion.
•Infection Assay: Some plates were infected with 1,000 RKN egg masses and others (20 plants)
were infected with 2,000 Meloidogyne incognita stage 2 juveniles (J2). J2 infections were examined
after one and seven days of exposure to the parasite.
•Staining methods: Acid fuchsin was used for staining root systems that were inoculated with the
nematodes. Since nematodes are translucent and difficult to see in plant tissues, acid fuchsin dye
allows for observation without excessively coloring the surroundings Arabidopsis thaliana plant
cells. The numbers of J2 nematodes, in the roots, after day one and seven days post infection
juveniles were averaged.
•RNA Isolation: Qiagen’ RNeasy plant RNA extraction protocol was used to isolate RNA from
mutant and Wild Type plants. Liquid nitrogen was used to stabilize plant tissue RNA in order to avoid
degradation and transcriptional induction.
• cDNA Synthesis: Total purified mutant RNA (1570 ng) of bothColWT (160ng/l) were used for
the starting template for reverse transcription to produce cDNA.
•RT-PCR: ColWT and mutant cDNA were tested to determine expression levels of an actin control
and Atpdr2 transporter genes. New England Biolabs quick-load Taq protocol was used for thirty five
cycles of PCR with annealing temperature at 52C. A second RT-PCR reaction as previously
described was done with adjusted annealing temperature at 50 C.
•Adult Nematode Identification: Leaves of infected plants, from 5 of the remaining plates were
removed and remaining media and roots were heated until liquefaction using EDTA. Acid stained
media contents were vacuum filtered. A screen cup collected roots while nematodes retained on filter
paper were examined with a dissection microscope and averaged. Female adult nematodes were also
counted and averaged.
Conclusions• The Atpdr2 mutant was heterozygous from the results of the DNA amplification , therefore Atpdr2 was not a mutant and was more
similar to the Columbia wild type.
• The Atpdr2 mutant transcript for the transporter gene was not shown on the cDNA amplification, but the actin was, showing that
the plant still has functioning proteins.
•The mutant had similar male and female nematode counts the wild type. This further supports that the mutant gene received was not
a mutant.
Results
The data displayed shows the genes DNA expression, the RNA transcript being produced by the mutant gene, the
number of juveniles, the number of males, and the number of female nematodes compared to the Columbia wild type.
Figure 1. T-DNA insertions of At4g15320 mutation (Atpdr2)
was not confirmed with a polymerase chain reaction (PCR)
gel analysis. Bands of Atpdr2 mutant (B) display slightly higher
expression intensities than Columbia Wild type (A) yet similar
migration rates were observed. Arabidopsis thaliana DNA from
mutant Col WT were isolated from the Syngenta Arabidopsis
Insertion Library (SAIL). Both DNA types were tested with
specific left border (LB) and reverse primers (RP) for the intact
gene of interest, and T-DNA (LB+ RP). A second LB primer
(LB2) was also tested in both DNA types .PCR reaction was
performed using 51C annealing temperatures for 35 cycles. On
an agarose (1%) gel with 1X TEA buffer, each lane was loaded
with Tri-Dye 2-log DNA ladder (10l), primers (0.5 l) DNA
(11.5 l) and Taq 2X Master Mix (12.5 l) Using gel
electrophoresis samples were visualized by ethidium bromide
stain contained within the Tri-Dye ladder which was later used as
a molecular weight standard for DNA mass approximation.
Bands showing higher intensity indicate reference points of
digested DNA fragments (C), ranging from 100bp to 10 kb.
Mutant and Col WT DNA fragments observed were
approximately 500bps and 124ng.
Figure 2. A second PCR reaction confirmed the
absence of T-DNA insertion in At4g15320
(Atpdr2) mutant. The reaction was set up at
previously described to retest for the T-DNA
insertion within the mutant. Annealing
temperatures were adjusted to 53C . The gel was
loaded as following; ladder (A) into lane 1, lane 2
did not contain a sample, lane 3 (LB+RP) and lane
4 (LB2+ RP). The absence of bands in both
mutant samples (C, D) indicate that the T-DNA
was not inserted into the mutant.
RNA Transcript Expression:
Confirmed Absence of T-DNA insertion:
Acknowledgements
We would like to thank our mentor Dr. Heather Marella for her guidance and support and our fellow classmates for their work on the
wild type data. Also, a special thanks to the rest of the BSU Biology Department.
LadderBlankCol WT
gene X
(1l)
Col WT
gene X
(2l)
Atpdr2
gene X
(1l)
Atpdr2
gene X
(2l)
1 2 3 4 65
Figure 4: A second reverse transcriptase PCR reaction was
preformed as previously described, with lowered annealing
temperatures (50C). To confirm the expression Atpdr2
transporter genes PCR samples were prepared with varying
amounts of cDNA (1l and 2l). Gel electrophoresis analyses
suggest multiple gene expression from both Col WT and mutant
line (lanes 1-4) in which two bands are observed in each. The
larger bands closest to the loading gel sites were the expected
observed genes while the smaller bands seen in both mutant
(lane 1) and Col WT (lane 2) appear to be expressed at increased
levels. Both Col WT and mutant A14g15320 (Atpdr2) show an
increase in intensity levels of gene expression in samples with an
both 1l and 2l of cDNA tested indicating sufficient transcript
was being made for a functional protein.
Figure 3. The bands observed indicate actin gene
transcript was expressed in At4g15320 (Atpdr2) mutant
and Col WT and confirmed by RT- PCR (lanes 4 and 6).
Atpdr2 transporter genes were not expressed by either DNA
types tested (lane 3 and 5). ColWT and mutant cDNA was
amplified using PCR and loaded on a 1% agrose for gel
electrophoresis to determine expression levels of an actin
control and Atpdr2 transporter genes. New England Biolabs
quick-load Taq protocol was used for thirty five cycles of
reverse transcript PCR with annealing temperature at 52C.
The following components were added to each PCR sample;
cDNA (1l) forward primer (1l), reverse primer (1l), 2X
Quick-load Taq Master Mix (12.5 l) and water (9.5l).
Samples were visualized by ethidium bromide stain contained
within the Tri-Dye ladder (lane 1). A second RT-PCR and gel
analysis was further used to re-test gene expression with
adjusted annealing temperature at 50 C.
Atpdr2
Actin
(1l)
Ladder Col WT
gene X
(1l)
Col WT
Actin
(1l)
Atpdr2
gene X
(1l)
Blank
1 2 3 4 65
LB2+RP
Col WT
LB+RP
Col WT
LP + RP
Col WT
LP+RP
Atpdr2
LadderLB+ RP
Atpdr2
LB2+RP
Atpdr2
Blank
1 2 3 4 65 7 8
Ladder Blank LB+RP
Atpdr2
LB2+RP
Atpdr2
1 2 3 4
5
Col WT increased J2 infection
B
C
0
4
8
12
16
20
24
1 day post-infection 7 days post infection
#JuvenilesinsideofRoot
ATPDR2 Mutant ColWt
Figure 5: Meloidogyne juveniles infection rate after 1 day and 7
days. The Atpdr2 mutant exhibited a reduced level of RKN
infection at both stages post infection. Meloidogyne incognita stage
2 juveniles (J2) were added to the Arabidopsis and allowed to infect
20 seedlings for one day and seven days. Acid fuchsin staining was
preformed to assess the number of nematodes infecting the roots
after day one and seven days of exposure to the parasite. The wild
type infection rate went from an average of 0 at day 1 to 17 at day
7. The mutant had 0 infections at day 1 and average nematodes was
2.1 at day 7.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Figure 6: Meloidogyne incognita average males for mutant
Atpdr2 and Columbia Wild type. The Atpdr2 mutant averaged 2
male nematodes per plate of Arabidopsis plated with a standard
error of 1.3. The Columbia wild type averaged 2.6 male
nematodes per plate of Arabidopsis with a standard error of 1.9.
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
Col WT ATPDR2 Mutant
EggMasses(%WT)
Figure 7: Meloidogyne average females for mutant Atpdr2 and
Columbia wild type.The Columbia Wild type averaged 107.1
females per infection of Arabidopsis with a standard error of 22.5.
The Atpdr2 averaged 82.2 females per infection with a standard
error of 25.0.
B
C
Figure 8: Acid fuchsin staining of Meloidgyne
incognita,a male root knot nematode stained with acid
fuchsin (A). Smaller J2 infective stage nematodes (B) can
also see here with the use of a dissecting microscope. The
Arabidopsis seedlings in wells of 1.5% bleach solution.(C)
with a 1:30 dilution of acid fuchsin stock in distilled water
(D).
A
A
D
C
Figure 7: Meloidogyne incognita infective stage
juveniles(J2), females, and egg sacs located through the
root systems of Arabidopsis thaliana. Female nematodes
(A), Nematode juveniles (B), and egg sacs (C) were
observed using a dissecting microscope.
B