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Gene silencing and gene expression inphytopathogenic fungi using
a plant virus vectorTiziana Masciaa,b,1, Franco Nigroa, Alì
Abdallaha, Massimo Ferraraa, Angelo De Stradisb, Roberto
Faeddac,Peter Palukaitisd,1, and Donato Gallitellia,b
aDipartimento di Scienze del Suolo, della Pianta e degli
Alimenti, Università degli Studi di Bari Aldo Moro, 70126 Bari,
Italy; bIstituto di Virologia Vegetale delConsiglio Nazionale delle
Ricerche, Unità Operativa di Supporto di Bari, 70126 Bari, Italy;
cDipartimento di Gestione dei Sistemi Agroalimentari e
Ambientali,Università degli Studi di Catania, 95123 Catania, Italy;
and dDepartment of Horticultural Sciences, Seoul Women’s
University, Seoul 139-774, South Korea
Edited by David C. Baulcombe, University of Cambridge,
Cambridge, United Kingdom, and approved February 7, 2014 (received
for review August 29, 2013)
RNA interference (RNAi) is a powerful approach for
elucidatinggene functions in a variety of organisms, including
phytopatho-genic fungi. In such fungi, RNAi has been induced by
expressinghairpin RNAs delivered through plasmids, sequences
integrated infungal or plant genomes, or by RNAi generated in
planta by aplant virus infection. All these approaches have some
drawbacksranging from instability of hairpin constructs in fungal
cells todifficulties in preparing and handling transgenic plants to
silencehomologous sequences in fungi grown on these plants. Here
weshow that RNAi can be expressed in the phytopathogenic
fungusColletotrichum acutatum (strain C71) by virus-induced gene
silenc-ing (VIGS) without a plant intermediate, but by using the
directinfection of a recombinant virus vector based on the plant
virus,tobacco mosaic virus (TMV). We provide evidence that a
wild-typeisolate of TMV is able to enter C71 cells grown in liquid
medium,replicate, and persist therein. With a similar approach, a
recombi-nant TMV vector carrying a gene for the ectopic expression
of thegreen fluorescent protein (GFP) induced the stable silencing
of theGFP in the C. acutatum transformant line 10 expressing GFP
de-rived from C71. The TMV-based vector also enabled C. acutatum
totransiently express exogenous GFP up to six subcultures and for
atleast 2 mo after infection, without the need to develop
transforma-tion technology. With these characteristics, we
anticipate this ap-proachwill findwider application as a tool in
functional genomics offilamentous fungi.
transfection | plant pathogen adaptation | host species jump
RNA interference (RNAi), RNA silencing, and gene quellingare
conserved processes in animals (1), plants (2), and fila-mentous
fungi (3), where they operate through diverse pathwaysbased on a
set of core reactions (4, 5). Briefly, these include thesynthesis
of a double-stranded RNA (dsRNA), which is recog-nized and diced
into 21- to 25-long dsRNA fragments by con-served ribonucleases of
the Dicer-like protein family. The smallfragments denoted small
interfering RNAs (siRNA) are thenloaded onto members of the
Argonaute protein family to forman RNA-induced silencing complex
that uses one of the twostrands of the siRNAs to direct RNA
degradation, translationalrepression, or DNA methylation of
homologous target genes(4, 5). These processes, commonly referred
to as RNAi, playa natural role in the regulation of endogenous gene
expression,development, and maintenance of genome integrity and
stabilityin reproductive cells, and in the protection against
transposableelements and viral infections. Besides its biological
role, RNAiwas recognized rapidly as a powerful approach for
elucidatinggene functions in a variety of organisms, including
phytopatho-genic fungi (5, 6). In these organisms, RNAi has been
inducedmainly with siRNAs derived from constructs expressing
dsRNAsor hairpin RNAs (hpRNAs) delivered through plasmids
orsequences integrated in fungal or plant genomes (7). In the
lattercase, the technique is referred to as host-induced gene
silencing(5) because it implies that siRNA molecules generated in
trans-genic plants expressing dsRNAs of fungal sequences
traffic
between the host and the infecting fungus. Conversely,
virus-induced gene silencing (VIGS) exploits viruses to deliver
sequencehomologous to a target gene fragment and trigger RNAi (8).
Itrequires the engineering of a viral genome to include fragmentsof
the target host gene or transgene to be silenced, the ability ofthe
recombinant virus to establish a systemic infection in the hostof
interest, and the silencing of the target gene by a
sequencehomology-dependent mechanism. VIGS is initiated by DNA
andRNA recombinant viral vectors as soon as their replication
pro-duces a dsRNA. This highly structured substrate is cleaved
intosiRNA by the enzymes of the RNAi pathway and the
signalamplified and spread throughout by an RNA-dependent
RNApolymerase coded by the host to down-regulate the expression
ofthe target gene (8). Thus, VIGS is a powerful reverse
geneticstool originally developed for plants, but new protocols and
viralvectors have expanded its utility also for functional genomics
infungi. For example, the barley stripe mosaic virus
(BSMV)-VIGSsystem has been used successfully for RNAi of specific
patho-genicity genes in Puccinia triticina by siRNA generated in
plantathrough infection of the BSMV vector expressing dsRNAs fromP.
triticina genes (9). Besides the complexity of some models
thatinclude transformation and handling of transgenic plants,
thestability of these dsRNA transcripts also has been questioned
infungi (10) and in viral vectors (11). When present on the
samestrand of viral nucleic acid, the engineered,
self-complementarysequences can enhance the effectiveness of
silencing but such in-verted repeat structures are often unstable
inside the viral genomes(11). Hence, we examined whether the plant
virus tobacco mo-saic virus (TMV) could be used for both gene
expression and
Significance
RNAi is used to elucidate gene functions also in
phytopatho-genic fungi, where it is expressed by hairpin RNAs
deliveredthrough plasmids, sequences integrated in fungal or
plantgenomes, or by RNAi generated in planta by a plant virus
in-fection. These approaches have drawbacks ranging from
in-stability of hairpin constructs to difficulties in preparing
andhandling transgenic plants to silence homologous sequences
infungi grown on these plants. Here we show that RNAi can
beexpressed in phytopathogenic fungi by direct transfection witha
plant virus-based vector and that the approach also can beused to
obtain foreign protein expression in fungi. This tech-nique could
find wider application for functional genomics infilamentous fungi
of biomedical and phytopathological interest.
Author contributions: T.M., F.N., and D.G. designed research;
T.M., F.N., A.A., M.F., A.D.S.,and R.F. performed research; F.N.
and M.F. contributed new reagents/analytic tools; T.M.,F.N.,
A.D.S., P.P., and D.G. analyzed data; and P.P. and D.G. wrote the
paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.1To whom correspondence
may be addressed. E-mail: [email protected]
[email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1315668111/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1315668111 PNAS | March 18,
2014 | vol. 111 | no. 11 | 4291–4296
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VIGS (8) by a direct infection in phytopathogenic fungi. It
wasknown that TMV could enter and persist in the mycelia of
thesoil-inhabiting phytopathogenic fungus Pythium sp. (now
placedamong oomycetes) (12), although no direct evidence for
viralreplication was provided. Similarly, Pythium arrehnomanes
couldbe transfected with TMV and tobacco necrosis virus (13).
Sincethen, the matter did not receive further attention, as the
interestfor viruses of fungi focused on true mycoviruses (14, 15).
In thisframework, a very recent study reported the infection of
intactmycelium of Sclerotinia sclerotiorum by the geminivirus-like
DNAmycovirus S. sclerotiorum hypovirulence-associated DNA virus
1,as a more efficient and easier to handle approach for
fungaltransfection (16).Here we show that RNAi can be expressed in
the phytopath-
ogenic fungus Colletotrichum acutatum by VIGS using a
recombi-nant vector based on TMV. We first provided evidence that
awild-type isolate of TMV was able to enter replicate and persist
incells of C. acutatum, strain C71 (C71). Subsequently, we used
arecombinant TMV vector, in which the ORF of the gene encodingthe
green fluorescent protein (GFP) was transcribed in C71 cellsfrom a
duplicate of the TMV coat protein (CP) subgenomic mRNApromoter and
demonstrated that the approach could be used toobtain foreign
protein expression in fungi. Finally we used theTMV-GFP construct
to abolish transcription and endogenous ex-pression of GFP in the
C. acutatum transformant line 10 expressingGFP (CATEF10)
transformant line of C71.
ResultsTMV Enters and Replicates in Cells of Colletotrichum spp.
A purifiedTMV suspension was added to a liquid culture of C71 and
my-celia were collected and examined at 5, 10, and 20 d after
in-cubation. Observations with a transmission electron
microscope
(TEM) on dips of mycelia of C71 treated with sodium
hypo-chlorite to eliminate virus particles adhering to hyphae
externallyshowed that TMV particles were present in mycelia already
after5 d of incubation and persisted therein at least for the
sub-sequent 15 d (Fig. 1 A and B). Ultrathin sections of
myceliacollected at 5, 10, and 20 d of incubation with TMV
showedaggregates of TMV-like rods (Fig. 1A) and membrane
vesiculationinside the C71 cells (Fig. 1B). These virus-like
particles and vesi-cles were not seen in the hyphae not exposed to
TMV (Fig. 1C),but were seen consistently in approximately 73% of an
average ofthe 2,000 hyphae collected and observed at each sampling
time.The presence of TMV particles in the C71 cells was confirmed
byin situ immunogold labeling (IGL), as abundant IGL signals
wereseen scattered in the cytoplasm at 5, 10, and 20 d of
incubationwith TMV (Fig. 1D), but not in control cultures (Fig.
1E). IGL didnot indicate any preferential localization for TMV
particles in thecytoplasm or cellular organelles, because of the
poorly resolvedcellular ultrastructure due to the IGL treatment.
Nevertheless,these results demonstrated that TMV particles entered
the C71hyphae and/or germinating conidia, probably through
woundscaused by either shaking or at structures or loci where
conidiagerminate and initiate hyphal development, colonized the
mycelia,and followed their growth up to 20 d of incubation. To
estimate thepercent of germinating conidia entered by virus
particles, C71 my-celia were sampled at 24 h after the addition of
a purified virussuspension to the culture in liquid medium. Twenty
mycelia sampleswere collected randomly, transferred individually
onto a solid sub-strate, and allowed to grow further for 1 wk.
Hybridization signalswith a TMV-specific probe were obtained
consistently from thetotal RNA extracts prepared from each of the
20 cultures (Fig.S1D). Because it was expected that after a 24-h
incubation inliquid medium no new conidia would be produced in the
culture,
Fig. 1. Modifications of cellular ultrastructure and
accumulation of TMV-like particles in hyphae of C71 collected at 20
d of incubation with TMV. Onlysamples collected at 20 d are shown
in this figure. Electron micrograph of a hypha of C71 exposed (A
and B) and not exposed (C) to TMV inoculum. In situlocalization of
TMV particles by immunogold labeling (IGL) in the same sample shown
in A, exposed (D) or not exposed (E) to TMV inoculum. Arrows in
Apoint to massive fibrous aggregates of TMV-like rods. (B)
Proliferation of the endoplasmic reticulum and dictyosomal vesicles
in hyphae of C71 coincubatedwith TMV. Insets show a closer view of
the rod-like aggregates in A, of membrane proliferation in B and of
IGL signals in D. Fibrillar materials outside the cellwall are not
formed by TMV particles because they were not targeted by IGL. They
probably represent fragments of the cell wall damaged by the
sodiumhypochlorite treatment as they were not seen in hyphae not
exposed to sodium hypochlorite treatment (Fig. S1C). (F) TMV
particles purified frommycelium ofC71 at 8 dpi with TMV. (G) TMV
particles purified from N. tabacum cv. Samsun nn. n, nucleus; cw,
cell wall. (Scale bars in A–D, 250 nm; F and G, Insets, 100
nm.)
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we estimate that soon after addition to the liquid medium
TMVentered 100% of either germinating conidia or wounded
hyphae.Quantitative estimates of total RNA extracted from groups
of
15 samples of the C71 mycelia, each group being collected at
5,10, and 20 d postinoculation (dpi) and determined by
dot-blothybridization with a TMV-specific probe, showed a sixfold
in-crease of TMV RNA accumulation from 5 to 20 dpi, indicatingviral
replication in C71 cells (Fig. S1E). A similar pattern wasobtained
also from total RNA preparations after hybridizationwith a
plus-sense DIG-labeled RNA probe, which detected
thereplication-specific, negative-sense strand of the viral RNA
(Fig.2A). A Northern blot analysis at 5 dpi with TMV provided
fur-ther evidence for viral replication in C71 cells by the
detection ofthe CP subgenomic RNA (Fig. 2B). However, the overall
accu-mulation of TMV RNA in C71 was much lower than in plants
ofNicotiana benthamiana at 5 dpi with TMV (Fig. 2B). Using thesame
approach, the analysis was extended to Colletotrichumclavatum,
isolate number 398854 from International MycologicalInstitute
(IMI), and Colletotrichum theobromicola, isolate F27,and in these
cases, TMV also entered, persisted, and replicatedin the mycelia of
both species (Fig. S1F).
TMV Does Not Alter Pathogenicity, Morphology, or Growth Rate
ofC71. Mycelia samples taken at 5, 10, and 20 dpi with TMV,treated
with sodium hypochlorite, crushed in phosphate bufferand rub
inoculated onto Samsun nn tobacco plants inducedchlorosis in
inoculated leaves by 5 dpi and systemic infection withappearance of
typical TMV-induced disease symptoms consist-ing of mosaic and leaf
blade deformation within 2 wk after in-oculation (Fig. S2A). The
presence of TMV RNA in both locallyand systemically infected leaves
of tobacco was verified by dot-blothybridization (Fig. S2D). Plants
rub inoculated with a mixtureobtained by crushing mycelia of C71
not exposed to coincubationwith TMV remained free of symptoms. The
virus was purified frommycelia of C71 collected at 8 dpi with TMV
and washed extensivelywith distilled water before purification
obtaining about 25 μg ofpartially purified virus suspension from 1
g of mycelia; i.e., a 15-foldincrease compared with the inoculum
applied to the medium. TMVparticles purified from C71 mycelia (Fig.
1F) were morphologi-cally undistinguishable from those routinely
obtained from plantinfection (Fig. 1G) and were infectious to
Nicotiana occidentalisand N. benthamiana plants inducing severe
local and systemic
symptoms (Fig. S2 B and C). These results demonstrate thatTMV
particles retained their original morphology and the abilityto
infect tobacco plants after entering and replicating in the cellsof
C71 mycelia. Moreover, TMV RNA was detected also in con-idia
harvested from TMV-infected C71 subcultured on plates atweekly
intervals for 2 mo (Fig. S1G). Thus, it appears that TMVpersisted
in intact conidia of C71, through which it could betransmitted to
other C71 generations during subculture.For pathogenicity tests, we
inoculated flowers of N. benthamiana
and wounded apple fruits of cv Golden Delicious with
suspensionsof conidia of C71 wild type or C71 transfected with TMV.
Both theoriginal wild-type and TMV-containing C71 cultures were
patho-genic on N. benthamiana flowers and apple fruit producing
typicalrot symptoms (Fig. S3 A and B). In these cases, the lesion
diameterinduced by the two cultures at 12 dpi was very similar
(Fig. S3C),suggesting that the presence of TMV in C71 neither
enhanced nordecreased the fungal pathogenicity in apple fruit.
Neither themorphology of C71 mycelia (Fig. S3D) nor the growth rate
of C71cultures on four different media (Fig. S3E) was altered
noticeablyby TMV infection. The involvement of
endopolygalacturonase(PG) in pathogenicity has been demonstrated
for several fungi,including C. acutatum (17, 18). However, TMV
infection of C71wild-type mycelia had no significant effect on PG
activity at 20 dpi(Fig. S3F).
Enhanced GFP Expresses Constitutively in Transformed C71.
Wetransformed the isolate C71 with a new binary plasmid vector
toexpress constitutively the enhanced GFP (eGFP) in Colleto-trichum
acutatum (pCATefGFP). This plasmid contained a con-struct in which
the expression of the eGFP was regulated by aconstitutive
translation elongation factor (TEF) promoter fromAureobasidium
pullulans, and the glucoamylase terminator fromAspergillus awamori.
A hygromycin B (HygB) resistance gene wasused as a selectable
marker. An average of 150 C71 transfor-mants per 105 conidia/mL
were obtained for the pCATefGFPconstruct and one of the
transformants, denoted CATEF10, wasselected for this study. The
expression of eGFP was confirmed byepifluorescence microscopy
analysis (Fig. 3 A and B), whereasthe results of sequencing and
restriction endonuclease double-digestion analysis revealed the
integration of the completeTEFefGFP cassette in the CATEF10 genome,
as a single in-sertion (Fig. S4A). The mitotic stability of the
integrated trans-gene was confirmed by more than 20 subcultures in
liquid andsolid media without HygB, and storage at −80 °C in water
con-taining 25% (vol/vol) glycerol. Pathogenicity tests conducted
onapple fruits did not show significant differences between
theCATEF10 line and the C71 wild-type isolate (Fig. S3C), al-though
the morphology of the respective mycelia was clearlydifferent (Fig.
S3D). The transgenic expression of eGFP in theCATEF10 isolate
induced also a ∼1.5-fold reduction in both thegrowth rate and the
PG activity, if compared with C71 wild-typeisolate (Fig. S3 E and
F). When viewed under TEM, cells ex-pressing GFP either
transgenically or ectopically showed elec-tron dense protein
aggregates (Fig. S5 B and D), which were notseen in C71 cells with
or without TMV infection (Fig. 1) or incells of CATEF10 with a
silenced eGFP phenotype (Fig. S5E).
The TMV-GFP Vector Down-Regulates eGFP Expression in CATEF10.The
inoculum for examining foreign gene expression and VIGSin the
mycelia was a recombinant, gene expression/VIGS vector,denoted
TMV-GFP, which had been established in N. occidentalisplants
inoculated with RNA transcripts synthesized from the bi-ologically
active plasmid pBSG1057-5. Sap from systemically in-fected leaves
of N. occidentalis emitting green fluorescence (Fig.S4C) was added
to the liquid medium during CATEF10 growth.The C71 wild-type
isolate challenged with the same sap was usedas a control. Mycelia
samples collected at 5, 10, and 20 dpi fromCATEF10 cultures
challenged with the TMV-GFP vector andobserved under an
epifluorescence microscope showed drasticallyreduced green
fluorescence in hyphae and conidia, starting from5 dpi and up to 20
dpi (Fig. 3 C and D). This result occurred in
Fig. 2. TMV enters and replicates in cells of C. acutatum. (A)
Determinationof the relative quantity (RQ) of TMV plus-sense RNA or
minus-sense RNA inpreparations of total RNA extracted from mycelia
samples of C71 collectedat 5, 10, and 20 d of incubation with TMV
estimated by dot-blot hybrid-ization with DIG-labeled minus- and
plus-strand RNA probes, respectively,against TMV RNA. Columns
represent the values of intensity of each spotquantified on the
basis of a standard curve obtained by fivefold dilutionseries of a
plasmid preparation at the initial concentration of 50 ng,
con-taining the same target sequence recognized by the probe. (B)
Abundanceof TMV RNA in 2 μg total RNA preparations extracted from a
N. benthamianaplant (T) and a C71 culture (C) at 5 dpi with TMV by
Northern blot hybridizationwith a DIG-labeled DNA probe. Arrows
indicate the positions of TMV genomic(gRNA) and coat protein
subgenomic (sgRNA) RNAs. Coat protein subgenomicRNA is not present
in RNA from purified virions. H, healthy N. benthamianaplant; T, N.
benthamiana infected by TMV; C, C71 infected by TMV; P, 200 ngRNA
extracted from a purified preparation of TMV.
Mascia et al. PNAS | March 18, 2014 | vol. 111 | no. 11 |
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all three samples collected at each sampling time, in contrast
toCATEF10 cultures challenged with sap from healthy N.
occi-dentalis, which remained green fluorescent (Fig. 3 E and F).
Toavoid any interference by siRNAs and dsRNAs resulting from
virusinfection in N. occidentalis and retained in plant sap used as
in-oculum, we also challenged CATEF10 cultures with a preparationof
particles of TMV-GFP (Fig. S5A) purified from N. occidentalisplants
showing bright fluorescence (Fig. S4C). Results from in-oculation
with a purified preparation of TMV-GFP were similar tothose
obtained with infected sap (Fig. S6 C and E).To demonstrate a
relationship between the reduction of green
fluorescence in CATEF10 cultures challenged with TMV-GFPand
RNAi, we analyzed samples of CATEF10 mycelia collectedat 5 and 10
dpi with TMV-GFP. The abundance of eGFP tran-scripts in nonsilenced
and silenced CATEF10 lines was esti-mated by quantitative PCR
(qPCR) of reverse-transcribed RNApreparations extracted from
samples of mycelia collected fromthe CATEF10 line at 5 and 10 dpi
with TMV-GFP. The resultsrevealed an almost complete
down-regulation in the eGFP geneexpression (Fig. 4A). As expected,
the expression of the GFPfrom the TMV-GFP vector also was highly
reduced if comparedwith the abundance of transcripts observed in
the C71 isolate at5 and 10 dpi with TMV-GFP (Fig. 4A). Effective
silencing of theGFP expressed from the TMV-GFP vector was shown
usinga primer pair specific for this sequence. Results shown in
Fig. 4Bdemonstrated that the expression of ectopic GFP in
CATEF10was reduced almost completely, similar to transgenic eGFP
(Fig.4A). Additional evidence for RNAi was given by total
RNApreparations hybridized with hydrolyzed DIG-labeled probes,
specific for the transgenic eGFP and for the CP gene of the
TMV-GFP vector, which showed the presence of siRNAs of
approxi-mately 21 nt specific for the eGFP gene and CP gene of
TMV-GFP (Fig. 4 C and D). These siRNAs were not seen in theCATEF10
line without TMV-GFP infection, providing indirectevidence that
ingress, replication, and expression of TMV-GFPtook place in cells
of CATEF10 and triggered a systemic RNAisignal that followed fungal
growth. Interestingly, as in plants,a siRNA population was produced
also against the CP gene ofthe TMV-GFP vector (Fig. 4D).
GFP Expression and Silencing Persists in Plated Cultures and in
PlantTissues. To test the stability of the CATEF10 silenced
phenotype13 monoconidial cultures of CATEF10 with a silenced
eGFPwere collected at 20 dpi from liquid medium and transferred
atweekly intervals on solid substrate for a further 45 d.
Fluores-cence was monitored at each passage, showing no reversed
si-lencing effects for the six subcultures (Fig. 3 G and H). Only
atthe seventh passage did 2 of the 13 monoconidial CATEF10cultures
again show the fluorescence signal. To assess whetherthe TMV-GFP
vector was still present and viable in eGFP-silenced CATEF10 cells,
total RNA was extracted from the sixthsubculture sample and
subjected to hybridization with a DNAprobe specific for the CP gene
of TMV-GFP. Fig. S4B showsa very weak signal of hybridization,
which parallels data fromqPCR (Fig. 4B); however, when mycelia from
this sixth subcul-ture of silenced CATEF10 were crushed in sodium
phosphatebuffer and rubbed onto leaves of N. benthamiana, N.
occidentalis,and tobacco, the plants remained free of symptoms and
TMV
Fig. 3. VIGS of GFP in CATEF10 transformant line upon ectopic
expression of GFP driven by the chimeric plant virus gene
expression/VIGS vector TMV-GFP.(A) Confocal microscopy showing
green fluorescence of the CATEF10 transformant line constitutively
expressing eGFP. (C) Epifluorescence microscopyshowing a culture of
CATEF10 with uniformly silenced eGFP at 10 dpi with TMV-GFP. (E)
Epifluorescence microscopy showing green fluorescence emittedfrom a
culture of CATEF10 at 10 d after incubation with sap of a healthy
N. occidentalis plant. Picture shows one of the three cultures
analyzed. (G) Epi-fluorescence microscopy showing a culture of
CATEF10 with still uniformly silenced eGFP after six subcultures on
solid medium (i.e., approximately 65 dpi withTMV-GFP). (I) Confocal
microscopy showing green fluorescence emitted by conidia of a
culture of C71 at 10 dpi with TMV-GFP. (K) Epifluorescence
microscopyshowing a culture of C71 at 10 dpi with wild-type TMV.
(M) Epifluorescence microscopy showing green fluorescence still
emitted by conidia of C71 infectedwith TMV-GFP after six
subcultures on solid medium (i.e., approximately 65 dpi with
TMV-GFP). (O) Epifluorescence microscopy showing a culture of
CATEF10with still uniformly silenced eGFP, recovered after
pathogenicity test in apple. A, C, E, G, I, K, M, and O and B, D,
F, H, J, L, N, and P show the same samplesviewed under UV and white
light, respectively. (Scale bars, 10 μm.)
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RNA was not detected in either inoculated or newly
emergingleaves up to 21 dpi. Collectively, these results
demonstrated thatRNAi activated in cells of CATEF10 line targeted
also TMV-GFP.Nonetheless, the eGFP knockdown in the CATEF10
transfor-mants still carried the eGFP gene insertion, because an
ampliconof the expected size was obtained using the primer pair
designed toamplify a 748-bp fragment from the eGFP sequence.Despite
the eGFP silencing, the overall morphology and my-
celial growth of both CATEF10 and the silenced CATEF10 werevery
similar, while retaining the original difference from the C71and
C71 + TMV-GFP isolates (Fig. S3D). A pathogenicity teston wounded
apple fruit (Fig. S3C) and olive seedlings (Fig. S7 Aand B) did not
show significant differences between the eGFP-silenced and
-nonsilenced CATEF10 lines. The green fluores-cence (or lack
thereof) from the transgenic fungi could not bedetected in either
infected apple fruit or olive seedlings due tofluorescence emitted
from the necrotized infected tissues. However,isolation of the
fungus from such fruit or leaves of olive seedlings 3wk
postinoculation and propagation on agar plates showed that theeGFP
in CATEF10 was still silenced (Fig. 3 O and P and Fig.
S7E),indicating that silencing of the transgene was maintained also
inliving tissues.Green fluorescent conidia and hyphae also were
seen scat-
tered in preparations of the wild-type C71 isolate
challengedwith the TMV-GFP vector (Fig. 3 I and J and Fig. S6 A and
B),but not in C71 cultures challenged with wild-type TMV (Fig. 3 K
andL). Expression of ectopic GFP in C71 cultures also was
demon-strated by qPCR with a primer pair specific for this GFP
sequence(Fig. 4B). The TMV-GFP–derived green fluorescence was
main-tained in C71 in 10 monoconidial cultures collected at 20 dpi
fromliquid medium, passaged on solid substrate six times at weekly
in-tervals, and monitored at each passage (Fig. 3 M and N). At
theseventh passage, 8 of the 10 isolates had lost the fluorescence
signal.
DiscussionThis study both provides unique information on the
ability ofa plant virus to infect and complete its replication
cycle in a plantpathogenic fungus and exploits a unique VIGS
protocol for fil-amentous fungi. Our results clearly show that TMV
is able toenter and replicate in cells of C. acutatum, C. clavatum,
andC. theobromicola, which may not be an exception, although it
has
neither been found nor probably searched for in nature.
C71hosting TMV replication retained overall morphology, growthrate
on different solid substrates, and PG activity, which isconsidered
one of the hallmarks for pathogenicity. These traitswere apparently
unaffected after several subcultures and storageup to 2 mo after
inoculation. Thus, TMV did not reduce thefitness of this fungus to
any noticeable extent. The most prom-inent alteration was at the
cell ultrastructure level, where accu-mulation of rod-like virus
particles was seen in the cytoplasm.More pronounced proliferation
of the endoplasmic reticulumand dictyosomal vesicles also were
observed (Fig. 1B and Fig.S5F) and by analogy with the
cytopathology of plants infected byTMV (19), they may represent the
sites for virus replication.Despite the ultrastructural changes and
accumulation of maturevirions, the overall load of TMV RNA in C71
tissues seemsrelatively poor if compared with that of infected
plants (Fig. 2Band Fig. S1E). In plants TMV distributes uniformly
during sys-temic infection, whereas we do not know whether and how
TMVmoves through C71 mycelia. Because growth of the mycelia
occursat the tips of the hyphae, we can speculate that TMV
distributesfrom one cell to the other during cell division at the
tips of hyphae.If this is the case, it would be expected that virus
could be presentprevalently in cells developed after virus entry,
which could resultin the relatively low estimates of viral RNA
loads detected inthis study.The observations that we described here
could be another
documented case of host species jumps for plant viruses
(20)without any apparent adverse effect for the virus fitness
afterexposure to a nonplant host. Replication of a few plant
viruseshas been documented in yeast (21), but it is previously
undoc-umented in filamentous fungi. We also showed that a
chimericplant virus could serve as a vector to abolish expression
ofa stably integrated and initially highly expressed transgene
infungal cells by the ectopic expression of sequences with
homol-ogy to the transgene. The obvious advantage of VIGS over
otherRNAi approaches in plants is that from the site of infection
theinoculum spreads systemically throughout the entire organism.The
situation is clearly different for the model proposed in thisstudy
in which viral inoculum is added to a culture of conidiaphysically
dispersed in the medium. However, following sap or pu-rified virus
inoculation of the liquid medium, TMV-GFP probably
Fig. 4. Reduction of GFP transcripts in CATEF10 transformant
line at 5 and 10 dpi with TMV-GFP. (A) Abundance of eGFP
transcripts in samples of CATEF10transformant line at 10 dpi
culture in liquid medium (CATEF10) challenged with sap from healthy
N. occidentalis compared with those detected in the sameline at 5
and 10 dpi with sap of N. occidentalis infected by TMV-GFP showing
nearly 100% reduction in the expression level of both transgenic
eGFP andectopic GFP carried by the TMV-GFP vector, as determined by
qPCR. C71, wild-type culture of C71 challenged with sap from
healthy N. occidentalis. C71 +TMV-GFP, accumulation of both the GFP
subgenomic RNA transcribed from TMV-GFP vector and the TMV-GFP
vector itself in wild-type C71 at 5 and 10 dpiwith sap of N.
occidentalis infected by TMV-GFP. (B) Amplification of GFP
expressed ectopically in C71 and CATEF10 cells upon infection with
TMV-GFP vectorby using selective primer pairs annealing to the coat
protein (CP) subgenomic promoter (ProFor) placed upstream the GFP
sequence and to a GFP fragment(GFPRev2) placed 137 nt downstream of
the ProFor primer in the sequence of TMV-GFP. The reduction of
TMV-GFP transcripts in CATEF10 +TMV-GFP at 5 and10 dpi provides
additional evidence for RNAi of viral GFP. (C and D) Detection of
siRNAs produced against GFP (C) and CP of TMV-GFP (D) in CATEF10
(CA10) at5 and 10 dpi with TMV-GFP (+TGFP) in preparations of total
RNA extracted from mycelia were hybridized with hydrolyzed probes
specific for GFP (C) and CPof TMV-GFP (D). M, size markers made by
19, 21, and 25 DNA oligonucleotides.
Mascia et al. PNAS | March 18, 2014 | vol. 111 | no. 11 |
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entered nearly 100% of CATEF10 cells as indirectly shown from
thecontrol C71 (Fig. S1D), so that the whole culture exhibited a
fulleGFP silencing phenotype, which was maintained still after
sixsubcultures. Interestingly, no infectivity of TMV-GFP was
detectedin plants back inoculated with crushed mycelia of such
subcultures,providing direct evidence that the silencing signal was
amplified,probably in each recipient cell, during fungal growth and
targetedboth the eGFP transcript and the TMV-GFP RNA. This, in
turn,implies also that the suppressor of RNA silencing encoded
byTMV would be ineffective in counteracting RNAi in fungal
cells.The observation that 8 of 10 monoconidial isolates of C71
lost thefluorescence expressed by TMV-GFP between the sixth and
theseventh subculture is consistent with this hypothesis. We
alsoprovided preliminary evidence that electron-dense
aggregatesaccumulated in cells of C71 upon infection with TMV-GFP
(Fig.S5 B and C) and that similar accumulation was also detected
inCATEF10 cells (Fig. S5D), but not in those with an eGFP
silencedphenotype (Fig. S5E).Compared with conventional gene
knockout strategies in
fungi, RNAi has several advantages and fewer drawbacks (22).One
major advantage is that RNAi can be induced transiently,overcoming
the need of permanent deletion of specific genes,which may be
lethal for the organism. In filamentous fungi, ef-ficient gene
silencing has been achieved by different approaches(7, 9, 10)
compared with which the VIGS strategy used in thisstudy is more
direct and easier to perform, as it obviates theplant-mediated
transferral of the silencing construct and is in-dependent of the
fungus asexual reproduction pathway.Like animal and plant viruses,
mycoviruses are also inducers,
targets, and suppressors of RNAi involved in the antiviral
de-fense response (23). In the case of Cryphonectria parasitica,
theRNAi-based response against hypovirus infection contributed
tohypovirus RNA recombination. In this regard, viral genome
in-stability represents an important obstacle to the use of
chimericmycoviruses as gene expression vectors and the discovery
ofa role for RNAi in promoting hypovirus RNA
recombinationpotentially hampers the use of chimeric mycoviruses as
vectors toinduce silencing in fungi (24). Also in this respect, our
approachto use VIGS vectors based on plant viruses could be a more
robust
tool. However, here we can anticipate that its use will need
somerefined insights to be more widely adopted. For example,
somefungi lack basic components of the RNAi silencing mechanism(10)
and the ability of viral vectors so far developed for VIGS inplants
to enter and replicate in fungal cells has to be investigatedcase
by case. For example, in an earlier study, an attempt to es-tablish
infection by BSMV in Gaeumannomyces graminis, Aur-eobasidium
bolleyi, and Pythium ultimum failed (25). Finally, theapproach
described in this study would also enable fungi totransiently or
chronically express other proteins without the needto develop
transformation technology for those fungi. This hasbeen shown by
the TMV-GFP infection in the C71 wild-typeisolate as the conidia
emitted green fluorescence and still con-tained the TMV-GFP vector
after six subcultures on agar plates.This paves the way for new
opportunities in the study of bothfungal physiology and
interactions with their hosts, applied tohuman, animal, and plant
mycology.
Materials and MethodsDetailed descriptions are provided in SI
Materials and Methods.
Fungi and Virus Isolates. The C71 strain of C. acutatum and the
isolates IMI398854 of C. clavatum and F27 of C. theobromicola were
grown on liquidmedium and inoculated with TMV or TMV-GFP. Mycelia
were collected at 5,10, and 20 dpi with TMV or TMV-GFP and treated
with sodium hypochlorite.
Transmission Electron Microscopy . Mycelia of C71 or CATEF10
collected at 5-,10-, and 20-d incubations with or without TMV or
TMV-GFP were used forthin sectioning. Immunogold labeling was
performed with a polyclonal an-tiserum raised against TMV.
Construction of the Binary Vector and Transformation of C.
acutatum C71. Theplasmid vector pCATefGFP construct containing the
eGFP sequence betweenthe constitutive TEF promoter from A.
pullulans and the glaA terminator fromA. awamori was electroporated
to competent cells of Agrobacterium tume-faciens AGL1 strain. For
the transformation, AGL1 cell lines were mixed withconidia of C71
and spread onto a sterile nitrocellulose membrane placed on topof
induction medium agar plates supplemented with hygromycin B for
selection.
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