Open Journal of Genetics, 2013, 3, 1-8 OJGen
http://dx.doi.org/10.4236/ojgen.2013.32A2001 Published Online July 2013 (http://www.scirp.org/journal/ojgen/)
RNAi technology targeting PbGP43 and PbP27 in
Paracoccidioides brasiliensis
Isaura Torres Gómez1,2, Orville Hernandez Ruiz1,3, Jose F. Muñoz1,2, Ana María Garcia1,
Angela Restrepo1, Juan G. McEwen1,4
1Unidad de Biología Celular y Molecular, Corporación para Investigaciones Biológicas (CIB), Medellín, Colombia
2Instituto de Biología, Universidad de Antioquia, Medellín, Colombia
3Grupo de Investigación en Biociencias, Facultad de Ciencias de la Salud, Institucion Universitaria Colegio Mayor de Antioquia,
Medellín, Colombia
4Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia
Received 7 May 2013; revised 7 June 2013; accepted 30 June 2013
Copyright © 2013 Isaura Torres Gómez et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Efficient technologies for gene silencing would be im-
portant to carry out functional analysis with P. bra-
siliensis genes, as well as for a better understanding of
the biology and pathogenesis of this pathogenic fun-
gus. Due to the fact that homologous recombination is
unusual in P. brasiliensis, the development of knock-
out isolates is currently non-feasible. The goal of this
work was to assess RNA interference (RNAi) techno-
logy as an alternative tool for gene silencing previous-
ly employed successfully in H. capsulatum. For this
purpose, we built different inverted repeat transgenic
hairpin constructs to down-regulate the PbGP43 and
PbP27 genes known to codify for two fungal immuno-
genic proteins that elicit a strong immune response
during experimental paracoccidioidomycosis. Using
the RNAi strategy, a reduction in the mRNA levels of
the PbGP43 and PbP27 genes was observed during the
first 20 days after selection; however, in the trans-
formed yeast cells, the gene silencing status proved
non-stable through the assay. We demonstrated that
electrotransformation was suitable to transform P.
brasiliensis yeast cells and integrate the hairpin con-
structions; nonetheless, gene silencing was not stable
along the experimental time. A detailed analysis of the
underlying molecular RNAi machinery may provide
further insights into the intracellular mechanism that
governs this reverse genetic tool.
Keywords: Paracoccidioides brasiliensis; Interference
RNA; Gene Silencing; PbGP43; PbP27; Gene
Expression
1. INTRODUCTION
RNA interference (RNAi), a natural mechanism con-
served all along evolution, has been implicated in gene
silencing in eukaryotic systems [1]. In addition, RNAi
participates in the regulation of genetic expression medi-
ated by certain classes of small endogenous RNAs such
as micro RNA (miRNA), which acts by using double
stranded RNA (dsRNA) homologous to the target se-
quence [2].
Due to the fact that generation of knockout isolates is
time consuming and requires sequential positive and ne-
gative selection steps, in order to enrich the desired re-
combination event, RNAi technology has rapidly become
one of the key methods in functional genomics studies,
and is used to block gene expression and create potential
phenotypes capable of yielding clues concerning the fun-
ction of these genes [2]. This technology has been suc-
cessfully used to obtain gene disruption in dimorphic
fungi, e.g. Histoplasma capsulatum [3] and Blastomyces
dermatitidis [4].
Paracoccidioides brasiliensis, a thermally dimorphic
fungus, is the etiological agent of paracoccidioidomyco-
sis (PCM), an important systemic, endemic mycosis in
Central and South America [5]. In this fungus, gene dis-
ruption methods are even more laborious and currently
seem unfeasible. This could be due to the presence of do-
minant illegitimate recombinant events (by non-homolo-
gous end-joining) overriding homologous recombination
[6]. In P. brasiliensis, RNAi could be employed as an
alternative method to transcriptional gene silencing me-
diated by sequence-specific mRNA depletion. The aim of
this work was to determine if RNAi strategy was a profi-
cient tool to down regulate both PbGP43 (a 43 kDa gly-

I. T. Gómez et al. / Open Journal of Genetics 3 (2013) 1-8 2
coprotein) [7] and PbP27 (a 27-kDa protein) [8] gene ex-
pression, two P. brasiliensis immunogenic antigens with
as yet unknown biological functions.
In this work, by means of bioinformatics analysis, we
demonstrated the presence of genes involved in the
RNAi route in the Paracoccidioides spp. genome. Our re-
sults indicated that RNAi strategy appears to be an effec-
tive method to integrate the RNAi hairpin constructions
in the genome of this fungus; however, the gene silenc-
ing status was not stable along the time in the isolates
evaluated.
2. MATERIALS AND METHODS
2.1. Strains and Culture Conditions
P. brasiliensis Pb339, a strain that produces high quanti-
ties of extracellular antigens, especially gp43 [9-11], and
p27 [8,12] during its parasitic phase was used in this stu-
dy. Yeast cell cultures and growth curves were performed
in BHI media supplemented with 1% glucose (Beckton
Dickinson and Company, Sparks, MD) at 37˚C with ae-
ration in a mechanical shaker and were routinely collec-
ted during the early exponential phase (72 - 96 h). Es-
cherichia coli DH5 grown at 36˚C in Luria Bertani (LB)
culture medium supplemented with appropriate antibiot-
ics, was used for cloning and plasmids propagation as-
says [13].
2.2. RNAi Orthologs in Paracoccidioides spp.
Genome
We performed a search in the Paracoccidioides spp. Ge-
nome references strains Pb18, Pb03 (P. brasiliensis) and
Pb01 (Paracoccidioides lutzii)
(http://www.Broadinstitute.org/annotation/genome/parac
occidioides_brasilien sis) of the putative homologous pro-
teins involved in Neurospora crassa RNA silencing [14]
using BLAST tools. Furthermore, we look for the key
domains involved in RNAi using profile hidden Markov
models (HMMs) with HMMER scan [15].
2.3. Molecular Cloning and Silencing Cassette
Construction
The RNAi plasmid pCR99 (Figure 1(a)) (provided by
Chad Rappleye, Washington University in St. Louis, Mi-
ssouri USA) was used to construct the silencing cas-
sette targeting P. brasiliensis PbGP43 and PbP27 (Fig-
ure 1(b)). H. capsulatum CBP1 promoter (889 bp) was
used to initiate transcription of RNAi targets, and the 733
bp intergenic region downstream of the CATB gene was
used as a transcriptional termination signal (T-catB). An
87 bp loop and either the approximate length of the dou-
ble-stranded were used in target region in the RNA hair-
pin. Primers were designed using P. brasiliensis strain;
sequence data available from
http://www.broadinstitute.org/annotation/genome). For
PbGP43 Pb18: PADG_07615 includes exon 1 (E1) and
exon 2 (E2); and for PbP27 Pb18: PADG_08402 (sup-
plementary Table Sl). To construct the PbGP43E1RNAi
cassette, a PbGP43 457-bp fragment was amplified from
genomic DNA and cloned in the opposite orientation into
pCR99 AscI-XhoI and AgeI-XbaI cloning sites; PbGP43-
(a) (b)
Figure 1. Hairpin RNAi constructions aimed at triggering gene silencing of PbGP43 and PbP27 in P. brasiliensis. A.
Map of the telomeric plasmid pCR99 used to trigger RNAi in P brasiliensis. H. capsulatum telomeres (TEL) were de-
scribed previously 16. The CBP1 promoter (889 bp) was used to initiate transcription of RNAi targets and the 733 bp
intergenic region down- stream of the CatB gene was used for transcriptional termination signals (T). This plasmid was
used to clone individually the inverted copies of target genes for PbGP43 exon 1 (PbGP43E1), exon 2 (PbGP43E2) and
PbP27, separated by a lacZ fragment to produce the RNA hairpin. Kan: kanamycin resistance; hph: hygromycin resis-
tance cassette. B. RNA hairpin constructs targeting PbGP43E1 (457 bp), PbGP43E2 (788 bp) and PbP27 (500 pb).

I. T. Gómez et al. / Open Journal of Genetics 3 (2013) 1-8 3
E2RNAi and PbP27RNAi cassettes were designed, am-
plified and constructed using the same strategy employed
to PbGP43E1RNAi cassette (Figure 1(b)). The primers
are described in supplementary Table S1, the sizes of the
target sequences were 788-pb and 500-pb respectively.
All PCR products were amplified using the Platinum
high-fidelity TaqDNA polymerase (Invitrogen, Carlsbad,
CA, USA).
2.4. P. brasiliensis Transformations and
Screening
The Pb339 strain was electrotransformed with PmeI-lin-
earized plasmid according to the protocol previously de-
scribed [16]. Briefly, P. brasiliensis yeast cells were
grown in BHI batch cultures to their exponenttial growth
phase with shaking at 36˚C, washed once with 10% man-
nitol, sterilized by filtration as electroporation solution.
Yeast cells were electrotransformed with 2 g of PmeI-di-
gested pCR99 constructions (PbGP43E1RNAi, PbGP43E2
RNAi and PbP27 RNAi) and an empty vector (PbEV) as
a control, in a Gene Pulser Electroporator (Bio-Rad, Her-
cules, CA), using the following conditions: capacitance
of 25 μF, resistance of 600 Ω and set voltage of 0.75 kV
[16,17]. Following transformation, cells were spread onto
selective BHI media supplemented with 100 μg/ml of hy-
gromycin B (Sigma, Aldrich, MO, USA). Selection plates
were monitored for colony forming ability at 37˚C for 15
to 20 days. The phenotypic stability of P. brasiliensis
transformants yeast cells was determined by analyzing
the stability of hygromycin B resistance [18]. Sixty indi-
vidual colonies were selected and subcultured in selec-
tive medium, (solid BHI containing 150 μgml hygromy-
cin B) each 5 days at 37˚C for three consecutive times
and then subcultured in liquid BHI containing 150 μgml
hygromycin B three times, RNA extraction was then
done and used in the RT-qPCR assay.
2.5. Molecular Detection of the Hygromycin
Resistance Gene (HPH)
Genomic DNAs from PbWt, PbEV, PbGP43E1RNAi,
PbGP43E2 RNAi and PbP27 RNAi transformants yeast
cells were isolated using the glass beads protocol de-
scribed by Van Burik (1998) [19]. In order to confirm the
presence of the hygromycin B resistance cassette, PCR
analysis was carried out to detect an hph 1000-bp ampli-
fication product using primers hphF (5’-AACTCACC-
GCGACGTCTGTCGA-3’) and hphR (5’-CTACACA-
GCCATCGGTCCAGA-3 ’). PCR amplification included
30 cycles of 1 min at 94˚C, for denaturation, 1 min at
68˚C for annealing, and 1.5 min at 72˚C for extension.
The reaction products were analyzed in 1% agarose gel
and visualized with ethidium bromide under UV light.
2.6. RNA Extraction, cDNA Synthesis and
Real-Time RT-qPCR Analysis
P. brasiliensis yeast cells were grown in liquid BHI sup-
plemented with glucose 1% and hygromycin B 150
g/ml at 37˚C and harvested after 5 days of growth.
RNA was obtained using the TRIzol® reagent according
to the manufacturer’s instructions (Invitrogen, Carlsbad,
CA, USA). Total RNA was treated with DNaseI (Invi-
trogen, Carlsbad, CA, USA) and tested for chromosomal
DNA contamination using conventional PCR for the β-
tubulin gene [20]. cDNA was synthesized using 1 µg of
total RNA and Superscript III reverse transcriptase accor-
ding to the manufacturer’s instructions (Invitrogen, Carl-
sbad, CA, USA). Real-time PCR (RT-qPCR) was per-
formed using Maxima® SYBR Green/Fluorescein qPCR
Master Mix, according to the manufacturer’s instructions
(Fermentas Maryland, USA). The CFX96 Real-Time PCR
Detection System (Bio-Rad, Headquarters Hercules, Ca-
lifornia, USA) was used to evaluate PbGP43 or PbP27
gene expression;
 -tubulin was selected as house keeping
gene [20]. Melting curve analysis was done after the am-
plification phase to eliminate the possibility of nonspe-
cific amplification or primer dimer formation. Folding
changes in mRNA expression were calculated using the
2∆∆CT formula, where ∆∆CT is the difference between
target and
 -tubulin genes [21]. Each experiment was car-
ried out in triplicate and the expression level was meas-
ured three times.
3. RESULTS
In P. brasiliensis Electrotransformed Yeast Cells,
PbGP43 and PbP27 Gene Silencing Achieved by
Using the RNAi System, Was Effective but Not
Stable during Time of the Experiments
Three different RNAi constructions were designed from
exon 1 and exon 2 from PbGP43 (PbGP43E1 RNAi,
PbGP43E2 RNAi) and one from PbP27 (PbP27 RNAi),
and transformed individually in PbWt yeast cells. Four-
teen transformants with reduced gene expression levels
were obtained by electrotransformation with the con-
structions PbGP43E1-RNAi (n: 6), PbGP 43E2-RNAi (n:
5) and PbP27-RNAi (n: 3) and selected after phenotypic
and mitotic stability tests (Table 1). The presence of the
RNAi cassette in the knock down strains and the yeast
cells transformed with PbEV was demonstrated by hph
gene amplification. This gene was no observed in PbWt
yeast cells (Figure 2).
In all yeast transformants cells, mitotic stability re-
mained stable in vitro cultures with continuous subcul-
tures in selective medium containing hygromycin B; in
parallel, all transformants were cultured several times in
non-selective medium; after this, all of them remained

I. T. Gómez et al. / Open Journal of Genetics 3 (2013) 1-8 4
Table 1. Genetic transformation of P. brasiliensis by electroporation using hairpin RNAi constructions.
Construct (2 μg) Colony number after phenotypic
stability test
Number of stable transformants
after mitotic stability test Mitotic stability (%)
PbGP43E1 RNAi 20 6 30
PbGP43E2 RNAi 31 5 16
PbP27 RNAi 33 3 9
Number of transformants obtained using 2 g of PmeI-linearized plasmid DNA; Mitotic stability of putative transformants was analyzed after 5 subcultures
on non-selective medium followed by plating on hygromycin B (150 g/ml).
PbGP43E1
PbGP43E2
PbP27
PbEV
PbWt
MW
1000 bp
PbGP43E1
PbGP43E2
PbP27
PbEV
PbWt
MW
1000 bp
Figure 2. Molecular analysis of the integration of the hph
resistance cassette into P. brasiliensis putative transfor-
mants.Wild type host strain, PbWt harboring the empty
vector (PbEV). Three putative transformants from the
RNAi hairpin constructions selected randomly were sub-
jected to PCR using the phosphotransferase gene (hph)
specific primers, hph-F and hph-R, in order to amplify a
1000 bp internal fragment of the hph gene MW: DNA mo-
lecular size marker.
their capacity to grow in the hygromycin B-containing
medium (Table 1).
After 20 days of growth in culture media, a decrease in
gene expression level ranging from 64% to 84% was
observed in PbGP43E1 RNAi isolates (Figure 3(a)), and
from 42% to 71% in the PbGP43E2 RNAi isolates (Fig-
ure 3(b)). In an equal manner, in the yeast cells trans-
formants that had been obtained using the PbP27RNAi
construction, a decrease in PbP27 gene expression level
ranging from 39% to 79%, was observed in comparison
with the expression levels in PbWt and PbEV (Figure
3(c)). However, after 45 and 60 days of growth in the
selective culture media, an increase in PbGP43 gene ex-
pression level in both PbGP43E1 RNAi and in
PbGP43E2 RNAi transformants was observed. Similarly,
an increase of PbP27 gene expression was observed in
PbP27RNAi yeast cells, indicating that in P. brasiliensis
electrotransformed yeast cells silencing had not been sta-
ble during the course of time.
BLAST searches using published data and available
genomes corresponding to P. brasiliensis Pb18, Pb03 and,
Pb01, as well as P. lutzii [22] (www.braodins titute.org)
were done. We identified all genes that participated in the
RNA silencing related with predicted function such RNA-
directed RNA polymerases (qde-1, sad-1, rrp-3), Argo-
naute-like (qde-2, Sms-2), Dicer-like (dcl-2, Sms-2) and
RecQ helicase-like (qde-3, RecQ-2) (Table 2). Further-
more, we found that the key protein domain of the RNAi
system in the proteins detected in the BLAST search,
indicating that the RNAi system could be used to down
regulate specific genes in P. brasiliensis (Supplementary
Figure S1).
4. DISCUSSION
Efficient technologies to achieve gene silencing could
help to undertake functional analysis of P. brasiliensis
genes and increase the understanding of the biology and
virulence attributes of this fungus, overcoming many of
the difficulties associated with traditional gene disruption.
RNAi does not depend on the homologous recombina-
tion machinery, making this strategy an attractive alter-
native to gene silencing [17].
In this study, we found that in Paracoccidioides spp,
there were homologous proteins involved in the RNA
silencing process suggesting the presence of the compo-
nents required for RNAi gene silencing thus supporting
the hypothesis that in this fungus such event should oc-
cur under natural conditions.
Our results show that the electroporation is an efficient
tool to introduce the linearized construction of RNAi in P.
brasiliensis yeast cells genome, as confirmed by the pre-
sence of the hph gene into the transformed cells. How-
ever, the reduced mitotic stability observed in the trans-
formant isolates would indicate that the fungus tends to
loose the hph fragment during continuous fungal growth
under non-selective pressure. As previously reported, the
efficiency of silencing depends upon several factors in-
cluding the length and structure of the hairpin construc-
tions. Rappleye et al., (2004) [17] have shown, that a
greater silencing effect was observed with a shorter loop
and a longer target gene sequence; these conditions were
used in our experiments but our results were different.
Currently, a gene silencing strategy based on anti-
sense-RNA (asRNA) has been efficiently employed in P.
brasiliensis to induce the knockdown of PbCDC42,
PbHAD32, PbAOX, and PbHSP90 [23-26]. Additionally,
based in our previous results using the antisense RNA
strategy targeting PbGP43 and PbP27 (unpublished), and

I. T. Gómez et al. / Open Journal of Genetics 3 (2013) 1-8 5
Figure 3. Gene expression levels of PbGP43 and PbP27 by RT-qPCR. (a) PbGP43 gene expression in
the PbWt, PbEV and the PbGP43E1 transformant yeast cells, selected after phenotypic and mitotic
stability. (b) PbGP43E2 RNAi gene expression in the PbWt, PbEV and PbGP43E2 RNAi transfor-
mants and (c) PbP27 gene expression in PbWt, PbEV and PbP27 RNAi transformants. All evaluations
were performed in the yeast cells after subculture for 20, 40 and 60 days. Low expression was ob-
served during the 20-day evaluation but not in those corresponding to the 45 and 60 days evaluations.
Gene expression levels obtained by RT-qPCR were normalized to the level of expression of the inter-
nal control gene TUB2 20.

I. T. Gómez et al. / Open Journal of Genetics 3 (2013) 1-8 6
Table 2. Presence of related RNA silencing genes in Paracoccidioides SP. Accession numbers of homologous genes founded in P.
brasiliensis (Pb18 and Pb03) and P. lutzii (Pb01) genomes, using BLASTX with predicted protein involved in the RNA machinery in
N. crassa [14].
Paracoccidioides
Predicted protein Neuros po ra c rassa Pb18 Pb03 Pb01
RNA-directed RNA polymerases qde-1 (NCU07534.1) PADG_07439.1 PABG_03975.1 PAAG_04185.1
sad-1 (NCU02178.1) PADG_03312.1 PABG_00852.1 PAAG_03813.1
rrp-3 (NCU08435.1) PADG_06286.1 PABG_06875.1 PAAG_03539.1
Argonaute-like, related to translation qde-2 (NCU04730.1) PADG_03108.1 PABG_00673.1 PAAG_03231.1
initiationfactors Sms-2 (NCU09434.1) PADG_00716.1 PABG_02302.1 PAAG_02052.1
Dicer-like, related to SFII-RNAse III dcl-2 (NCU06766.1) PADG_07189.1 PABG_05105.1 PAAG_00072.1
Ribonucleases of the carpel factory Sms-3 (NCU08270.1) PADG_05567.1 PABG_04917.1 PAAG_02421.1
RecQ helicase-like, related to Bloom’s qde-3 (NCU08598.1) PADG_03738.1 PABG_01669.1 PAAG_01087.1
and Werner syndrome helicases RecQ-2 (NCU03337.1)73 PADG_08306.1 PABG_07570.1: PAAG_07608.1
the results obtained in this work using RNA interfer-
ence strategy, we conclude that the non-stability in gene
silencing is due to strategy and transformation methodo-
logy employed to down regulate specific gene. However,
it will be important to explore an alternative gene silenc-
ing strategy such as RNAi, in order to diminish the time
required for obtaining transformants with the desired
phenotype, and achieve higher transformation efficiency.
For the above reasons, and taking into account that RNAi
machinery is present in the Paracoccidioidesspp ge-
nomes, and that the strategy has been employed efficient-
ly in H. capsulatum, we conclude that RNAi is an easier
and faster tool than asRNA for gene silencing and has all
potentialities for the study of many functional genes in P.
brasiliensis. However further studies need to be conduc-
ted for the synthesis of new and more stable construc-
tions based on RNAi technology. Presently, use of the
transformation system mediated by A. tumefaciens, which
could show best results in P. brasiliensis gene silencing
[23-26].
This is the first report using electrotransformation of
hairpin construction based on RNAi technology targeting
two important P. brasiliensis antigens (gp43 and p27). A
detailed analysis of the underlying molecular RNAi ma-
chinery may provide further insight into the intracellular
mechanism that governs this reverse genetic tool. Present
results provide an initial framework for further studies of
virulence factors in fungi such as P. brasiliensis, in which
the genetic manipulation represents a challenge.
5. ACKNOWLEDGEMENTS
This work was supported by Colciencias project No 221340820447.
Support was also obtained from the Corporación para Investigaciones
Biológicas (CIB) and the Universidad of Antioquia through the fund
“Sostenibilidad 2010-2011”. COLCIENCIAS National Doctoral Pro-
gram supported Isaura Torres.
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APPENDIX
Supplementary Digital Content S1
Table S1. Primers used to construct the RNAi cassettes targeting P. brasiliensis PbGP43 and PbP27.
Target gene (hairpin size) Primer name Primer Sequence 5’-----------3’
PbGP43E1-AscI-F AGGCGCGCCTTTAGTTCTCTCAACCTGGC
PbGP43E1-XhoI-R GTCTCGAG CACCATGGAGATCGATGACG
PbGP43E1-XbaI-F CGTCTAGATTTAGTTCTCTCAACCTGGC
PbGP43 E1 (457-bp)
PbGP43E1-AgeI-R GTACCGGTCACCATGGAGATCGATGACG
PbGP43E2-AscI-F AGGCGCGCCTCCCGGGTTCTCAAAACGGC
PbGP43E2-XhoI-R GTCTCGAGTCACCTGCATCCACCATACT
PbGP43E2-XbaI-F CGTCTAGATCCCGGGTTCTCAAAACGGC
PbGP43 E2 (788-bp)
PbGP43E2-AgeI-R GTACCGGTTCACCTGCATCCACCATACT
PbP27-AscI-F AGGCGCGCCTTCCGACGAGCTGAAAACTG
PbP27-XhoI-R GTCTCGAGACAGCGTGCATTTGATCTT
PbP27-XbaI-F CGTCTAGATTCCGACGAGCTGAAAACTG
PbP27 (500-pb)
PbP27-AgeI-R GTACCGGTATGATATCACGAATATCGGT
Figure S1. RNAi silencing in Paracoccidioides spp.: Key protein domains. Nine genes identified in the P. brasiliensis
genome (strain Pb18) using BLAST showing the presence of protein domains involved in the RNAi silencing.
Argonaute-like proteins (a,b) showing the PIWI (green) and PAZ (blue) domains. Dicer-like proteins (c,d) presenting the
Ribonuclease III (orange) and double stranded RNA binding (blue pentagon) domains. Helicase conserved C terminal
domain (blue circle); in d, e and f, DEAD/DEAH box helicase domain (green pentagon); in the Helicase-like protein (e,f)
and the RNA dependent RNA polymerase domains (orange) in g, h, and i.