Experimento molecular que sugiere que la tolerancia de Ulmus minor a la grafiosis está relacionada a la expresión diferencial de algunos genes como aquellos relacionados con GO:000960

Collada Collada, Maria Carmen

Domínguez, Jorge

Gil Sanchez, Luis Alfonso

Martin Garcia, Juan Antonio

Medel, David

Perdiguero Jimenez, Pedro

Sobrino-Plata, Juan

Venturas, Martín David

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Dissecting the molecular responses potentially involved in the tolerance of two Ulmus minor

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DISSECTING THE MOLECULAR RESPONSES POTENTIALLY INVOLVED IN THE TOLERANCE OF 
TWO ULMUS MINOR GENOTYPES DURING OPHIOSTOMA NOVO-ULMI COLONIZATION 
Pedro Perdigueroa,b, Martin Venturas b,c, Juan Sobrino-Platab, David Medelb, Jorge Domínguezb, Juan Antonio Martínb, Luis Gilb, Carmen Colladab 
aiBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal bGENFOR. Grupo de investigación en Genética, Fisiología e 
Historia Forestal, Universidad Politécnica de Madrid. Madrid, Spain cBiology Department, University of Utah, Salt Lake City, USA 
Dutch elm disease (DED) is a vascular wilt disease caused by two fungi, 
Ophiostoma ulmi and the more aggressive, Ophiostoma novo-ulmi. The 
outbreak of two pandemics during the last century severely affected North 
American and European elm populations. Nowadays, numerous trees are 
still dying due to the difficulties to control this highly virulent disease. The 
Spanish elm breeding programme has obtained seven highly tolerant native 
U. minor genotypes that are registered as forest reproductive material [1]. 
<+H 
Ophiostoma 
ulmi 
Ophiostoma 
novo-ulmi 
First 
pandemic 
Second 
pandemic 
1910 30 
Adapted from Brasier (2000) 
50 70 
1 
90 Year 
A selection of unigenes from a biotic stress library of Ulmus minor [2] 
was included in the microarray design (Agilent 8 x 60K, Agilent Technologies, 
CA, USA). A total of 1,681 unigenes significantly modified their level of 
transcripts in at least one sampling point in one genotype during Ophiostoma 
novo-ulmi infection. 300 genes were exclusively identified in AB-AM2.4, 167 
genes in M-DV2.3 and 50 genes in both genotypes 
Top upregulated genes exclusively found in AB-AM2.4 genotype at short and long-term after 
inoculation with Ophiostoma novo-ulmi 
IDJSOTIG 
isotig 18444 
isotig08555 
isotig21148 
isotig07125 
isotig08310 
isotig 13236 
isotig20653 
isotig13410 
isotig02505 
isotig02150 
isotig07680 
isotig19684 
isotig 16541 
isotig 10720 
isotig 15975 
isotig12538 
isotig04982 
isotig 18079 
isotig05901 
contig08923 
isotig04660 
isotig19859 
isotig14781 
isotig 18662 
isotig01174 
isotig 13783 
isotig 13774 
isotig 16863 
isotig 13964 
isotig19668 
DESCRIPCIÓN BLAST2GO 
acidic endochitinase-like 
udp-glucoronosyl udp-glucosyl transferase family protein 
thioredoxin-like protein 
basic form of pathogenesis-related protein 1 
(-)-isopiperitenol (-)-carveol mitochondrial-like 
... NA— 
udp-glycosyltransferase 75d1-like 
probable glutathione s-transferase 
probable lrr receptor-like serine threonine-protein kinase at1g53430-like 
ethylene-responsive transcription factor rap2-12-like 
at5g56980 mhm17_10 
cationic amino acid transporter 1-like 
probable s-adenosylmethionine-dependent methyltransferase at5g38100 
light-inducible protein cprf2-like 
multiple c2 and transmembrane domain-containing protein 1-like 
cellulose synthase-like protein g3-like 
arabinogalactan peptide 20-like 
transcription factor bhlh36-like 
conserved hypothetical protein 
cell wall-associated partial 
cell wall-associated hydrolase 
outer envelope pore protein 16- chloroplastic-like 
ethylene-responsive transcription factor erf109-like 
1-phosphatidylinositol- -bisphosphate 
probable xyloglucan endotransglucosylase hydrolase protein 23-like 
abscisic acid receptor pyl4-like 
u-box domain-containing protein 21-like 
homeobox-leucine zipper protein athb-12-like 
e3 ubiquitin-protein ligase pub23-like 
hypothetical protein POPTR_0019s10350g 
D1 D3 
-1,64 
-2,59 
1,86 
2,08 
1,91 
2,10 
2,78 
2,50 
2,40 
2,35 
2,32 
2,16 
2,07 
D7 
2,14 
1,95 
1,74 
-1,73 
D14 
3,68 
2,59 
2,10 
D21 
14,73 
9,59 
7,30 
7,07 
5,94 
5,63 
4,99 
4,98 
4,60 
4,45 
4,33 
4,00 
3,90 
3,88 
3,80 
3,78 
3,74 
3,73 
3,27 
Year 
Time 
M-DV2.3 
AB-AM2.4 
M-DV1 
2011 2012 
% Wilting 30 d.p.i 
1.7 ± 4.8 
36.5 ± 4.8 
82.3 ± 4.8 
3.1 ± 7.7 
34.8 ± 7.7 
58.9 ± 8.9 
2011 2012 
% Wilting 60 d.p.i 
1.4 ± 4.9 
31.9 ± 4.9 
86.7 ± 4.9 
1.7 ± 5.4 
28.6 ± 5.4 
80 ± 6.3 
2011 2012 
% Wilting 90 d.p.i 
3.3 ± 5.4 
32.3 ± 5.4 
87.7 ± 5.4 
3.5 ± 5.4 
26.9 ± 5.4 
81.7 ± 6.2 
Three Spanish elms with contrasted tolerance to 
DED were selected for this study (Table above 
shows canopy wilting percentage in a previous 
experiment). Inoculation was carried out about 15-30 
days after full leaf development. Symptoms were 
visually assessed during four months after 
inoculation by estimating the percentage of leaf 
wilting (shown in Table bellow the image). Inoculated 
plants from susceptible genotypes showed the first 
symptoms in the crown at 14 days post inoculation, 
reaching 10-15% of wilting leaves. By 21 days after 
inoculation, tolerant genotypes inoculated with 
Ophiostoma novo-ulmi showed a low percentage of 
wilting (10-15%) whereas susceptible genotypes 
reached values exceeding 50%. 
Differentially expressed genes follow two main trends; increase or decrease in 
transcript levels. Graphics on the left show the average trend for each genotype. An 
enrichment analysis indicated an increase of Gene Ontology (GO) terms related to 
“response to biotic stimulus”, “response to stress” or “kinase activity” in genes 
upregulated (blue bars) whereas downregulated genes showed significant 
enrichment in GO terms related to anatomical structure morphogenesis, cell growth 
or cell differentiation (red bars). 
Significant increases 
in level of transcripts 
of specific defence 
genes or transcription 
factors were identified 
specifically in tolerant 
genotypes. These 
results suggest that U. 
minor tolerance to O. 
novo-ulmi is related to 
the differential 
expression of these 
genes. 
Top upregulated genes exclusively found 
inoculation with Ophiostoma novo-ulmi 
in M-DV2.3 genotype at short and long-term after 
IDJSOTIG 
isotig00311 
isotig04987 
isotig20904 
isotig09369 
isotig20747 
isotig17060 
isotig08725 
isotig17614 
isotig06888 
isotig01332 
isotig04713 
isotig18152 
isotig21152 
isotig17069 
isotig18160 
isotig06364 
isotig18411 
isotig20057 
isotig20807 
isotig10818 
DESCRIPCIÓN BLAST2GO 
hypothetical protein POPTR_0002s22940g 
two-component response regulator arr9-like 
protease inhibitor seed storage lipid transfer family protein 
glutaredoxin family protein 
two-component response regulator arr5-like 
protein glutamine dumper 3-like 
expansin a1 
heavy metal-associated isoprenylated plant protein 26-like 
probable rna-dependent rna polymerase 1 
laccase-14-like 
PREDICTED: uncharacterized protein LOC103436664 
probable calcium-binding protein cml10-like 
- N A -
probable xyloglucan endotransglucosylase hydrolase protein 23 
copper transporter 1-like 
ud p-g lycosyItransferase 85a2-like 
transcription factor bhlh30-like 
u-box domain-containing protein 19-like 
hypothetical protein POPTR_0001s10440g 
two-component response regulator-like prr95-like isoform x3 
D1 
1,91 
-2,00 
D3 D7 
-1,60 -1,65 
1,73 
2,94 
2,30 
2,29 
2,00 
1,96 
1,88 
1,83 
1,80 
D14 
2,07 
2,63 
2,31 
2,26 
2,04 
D21 
2,30 
2,06 
1,94 
1,94 
1,84 
1,79 
1,79 
1,77 
References 
[1] Martín J, Solla A, Venturas M, Collada C, Domínguez J, Miranda E, Fuentes P, Burón M, Iglesias S, Gil L, (2015) Seven Ulmus minor clones tolerant 
to Ophiostoma novo-ulmi registered as forest reproductive material in Spain, iForest - Biogeosciences and Forestry 8, 172-180. 
[2] Perdiguero P, Venturas M, Cervera MT, Gil L, Collada C. (2015). Massive sequencing of Ulmus minor's transcriptome provides new molecular tools 
for a genus under the constant threat of Dutch elm disease., Frontiers in Plant Sciences, 6:541. 
Acknowledgements 
This research was funded by the Ministerio de Agricultura, Alimentación y Medio 
Ambiente (MAGRAMA) and by the Spanish National Research Plan (AGL2012-
35580). We would also like to express our gratitude to the Spanish Elm Breeding 
and Conservation Programme. 


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Dissecting the molecular responses potentially involved in the tolerance of two Ulmus minor

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