1. Introduction
• The Phytophthora genus has been studied and used to
elucidate many important genetic patterns and
molecular traits of oomycetes. Oomycetes are a class of
fungus-like organisms within the kingdom
Chromalveolata (Tyler 2006). Fungi and oomycetes share
many morphological features (mycelium, spores, etc.).
However, oomycetes are thought to be more closely
related to plants than to true fungi due to similarities in
cell wall structure of the two groups (Latijnhouwers
2003).
• Within the Phytophthora genus are many examples of
deadly plant pathogens. One of the most impactful
species worldwide is the soybean pathogen, P. sojae. This
species causes soybean root rot in susceptible hosts, with
crop losses totaling in the millions annually (Tyler 2007).
The study of P. sojae genes as they relate to
pathogenicity is an important way to gain insight into
new methods of control of this and other Phytophthora
species.
• Polysaccharide lyases (PLs) are a superfamily of enzymes
used by P. sojae to hydrolyze the carbohydrates in the
plant cell wall, thereby, degrading the host’s cell walls.
The PL superfamily does this via cleavage of
polysaccharide chains containing uronic acid (Lombard
2010). PL families are varied and their presence is
conserved in many Phytophthora spp. analyzed thus far.
In this study, we analyzed the expression of a family of
PL sequences in infected plant tissue and Phytophthora
sojae mycelium grown in vitro.
Results and Discussion
• Infection of 5 Williams 82 soybean plants with P.
sojae R4 yielded 5 useable leaf samples per time
point: 12, 24, 36, and 48 hours post infection (hpi)
(Figure 1).
• RNA extractions yielded the streak of bands
indicative of total RNA with no degradation for the
12 and 24 hpi soybean samples.
• RT- PCR yielded useable cDNAs for 13 out of 15 total
sequences for the mycelial reference sample, as
confirmed by gel electrophoresis (Figure 2). The 12
hpi time point yielded 9 0f the 15 sequences, and
the 24 hpi time point yielded only 4. Those
sequences not included in these numbers (except
for PSPL14 and 15) yielded unreliable results that
were not suitable for qPCR analysis. This may be
based on a concertation of cDNAs or RNA in the
preceding experiments that was too low.
• qPCR threshold cycle data for those sequences that
yielded useable cDNAs at both time points were
collected; statistical calculations using ΔCT and
ΔΔCT values yielded the expression fold changes
observed in Figure 3. The time needed to work
through the minutiae of the procedure limited the
amount of results that were obtained
Figure 2: The gel picture following the RT-PCR procedure for the
12 hpi samples for the following sequences and negative controls:
PSPL 1 and 1- (lanes 4 and 5), 2 and 2- (lanes 6 and 7) 9 and 9-
(lanes 8 and 9) and 12 and 12- (lanes 10 and 11). Lane 2 contained
the 1 kb DNA ladder.
Materials and Methods
Primer Design: For the sequences in P. sojae that yielded negative control expression at To, primers were re-designed using the Primer-BLAST website. Primers were optimized based on the recommendations given in the Bio-Rad iQ5 Real-Time PCR handbook. Primer sequences will be designed as to anneal to regions of each gene so that a
portion (~80-150 bp) of the sequence would be amplified during later Polymerase PCR and qPCR experiments.
Soybean infection/sample collection: ~4 week old Williams 82 soybean was used, along with P. sojae (strain R5). A small incision was made below the plant’s hypocotyl, and an agar plug was inserted. At each timepoint, a sterile razor blade was used to cut the leaves above the incision site. The leaves were flash frozen and stored at -80 °C
(described in Ospina-Giraldo et al., 2007).
RNA Extraction: The QIAGEN RNeasy Plant Mini Kit extraction kit was used for the RNA extraction procedure, and the included protocol in the manual was followed. This included the optional on-column DNase digestion, to eliminate genomic DNA that would interfere with further procedures. A subsequent agarose gel electrophoresis
was run to visualize the results. For mycelia, pea broth culture samples were filtered in a Buchner funnel with filter paper, and the resulting tissue was used in the same way as infected plant tissue.
RT-PCR: The QIAGEN QuantiTect Reverse Transcription kit was used for the RT-PCR, and the included protocol in the manual was followed. The procedure allowed the creation of cDNA from extracted RNA at the four timepoints, usable for further testing
PCR and Gel Electrophoresis: PCR analysis was performed on the cDNA and negative controls obtained from the RT-PCR to test the alignment of the cDNAs to specially designed primers ( the negative controls, being RNA, would not have annealed to the primers). 0.5 μL of each of the PSCS15F/R primers designed by Lauren Hinkel were
used, along with 1 μL cDNA (or negative control product), 0.5 μL dNTPs, 5 μL Taq buffer, 0.5 μL Taq polymerase, and 12 μL dH2O. The thermocycler settings were: 1 cycle at 94 °C for 5 mins, 35 cycles at 94 °C for 30 s, 63 °C for 1 min, and 72 °C for 20 s, and 1 cycle at 72 °C for 5 mins. A 1 % agarose gel (40 mL 1xTAE, .4 g agarose, 500 μL EtBr) was
run at 80 volts for 40 mins for visualization.
qPCR Analysis: qPCR reactions used the QIAGEN QuantiScript SYBR Green PCR Kit. They featured the following amounts of specified reagents, creating 2 25µL replicates: 25 µL SYBR Green Master Mix, 18 µL dH2O, 5 µL cDNAs or negative control product, 1 µL PSCS15R primer, 1 µL PSCS15F. The recommended thermocycler settings found in the
kit manual were used, with Tm varying from 64 to 64.7 C for 12 to 7s for successful trials. Threshold cycle values were recorded and used to report the ΔCT and ΔΔCT values reported above.
Literature Referenced
Erlich, HA. PCR technology: Principles and applications for DNA amplification. Stockton press, 1989.
Kamoun, Sophien. "Molecular genetics of pathogenic oomycetes." Eukaryotic cell 2.2 (2003): 191-199.
Latijnhouwers, Maita, Pierre JGM De Wit, and Francine Govers. "Oomycetes and fungi: similar weaponry to attack plants." Trends in microbiology 11.10 (2003): 462-469.
Lombard, Vincent, et al. "A hierarchical classification of polysaccharide lyases for glycogenomics." Biochemical Journal 432 (2010): 437-444.
Tyler, Brett M., et al. "Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis." Science 313.5791 (2006): 1261-1266.
Tyler, Brett M. "Phytophthora sojae: root rot pathogen of soybean and model oomycete." Molecular plant pathology 8.1 (2007): 1-8.
Acknowledgements
The author would like to thank Professor Manuel Ospina-
Giraldo for his lab expertise, Samantha Gleich and Cassidy
Maddison for their procedural assistance, and Phillip
Auerbach for technical assistance
Brandon Ross and Manuel Ospina-Giraldo
Biology Department, Lafayette College
EXPRESSION ANALYSIS OF POLYSACCHARIDE LYASE SEQUENCES DURING INFECTION OF
SOYBEAN BY PHYTOPHTHORA SOJAE R4
Figures 3 and 4: Expression fold change, based on a log10 scale for the indicated timepoints. ΔCT vales correspond to the difference in the average threshold
cycle subtracted from the same value found for actin. ΔΔCT values correspond to the ΔCT values for each timepoint subtracted from the same value found
for P. sojae mycelium (values not shown). Both were used in the calculation of expression fold change. For the sequences that yielded useable data, all are
see to be downregulated from 12 to 24 hpi but still upregulated between mycelium and 24 hpi.
Objectives
• Develop appropriate negative controls for the
study
•Use RNA Extraction, Reverse
Transcription PCR(RT-PCR, to obtain
cDNAs) and qPCR analysis relative to
actin and basal mycelium levels to study
expression
Conclusions
• Based on the lack of expression fold change
for T0, the designed primers were specific for
the selected genes.
• There is a noticeable difference in the levels
of expression of selected sequences at the
time points shown in Figures 3 and 4.
Additionally, all 24 hpi time point data
showed lower levels of expression than 12 hpi
but not mycelium.
Figure 1: The effects of the infection process at 24 hpi on a
Williams 82 soybean plant. Discoloration and leaf spots can
be seen on the upper two leaves, along with some leaf tissue
collapse on the lower leaf. These are classic symptoms of
plant response to pathogens .
.
1 2 3 4 5 6 7 8 9 10 11
2 3 4 5 7 10 11 12 13
12 hpi 493 30.8 108 60.8 136 35.3 105 314 3752
1
10
100
1000
10000
100000
FoldChangeinExpression[log10]
Figure 3: Fold Change in Expression of
Selected PSPL Sequences (12 hpi)
5 7 11 13
24 hpi 5.302478 61.96292 41.06963 2154.793
1
10
100
1000
10000
FoldChangeinExpression[log10]
Figure 4: Fold Change in Expression of
Selected PSPL Sequences (24 hpi)