Sistemas redox en Botrytis cinerea:

Impacto en el Desarrollo y Virulencia

AREA:
Interacción Molecular Planta Patogeno
DOCENTE:
-Jose M. Soto Heredia

ALUMNOS:
- Diego Fernando Luján Ajalcriña (20200601)
- Miguel Maturrano
INTRODUCCION
Tabla 1. Especies reactivas de oxígeno (34). Fuente: Review Cellular
defenses against damage from reactive oxygen species. Yu P. 1994
Las plantas liberan ROS como estrategia de
defensa como parte de la respuesta
hipersensible (HR).

B. cinerea depende de esta muerte celular

programada, parece requerir la participación
activa del huésped para lograr la virulencia total
(Govrin y Levine 2000; van Kan 2006).

La exposición de moléculas biológicas a ROS

también conduce a daños moleculares severos.

El hongo debe garantizar una rápida renovación

de ROS para mantener el equilibrio entre los
equivalentes oxidantes y reductores, que
constituyen el estado redox intracelular (Jones
2008; Torres y Dangl 2005).

Todos los organismos productores de ROS

pueden evitar niveles elevados de ROS
mediante sistemas de eliminación eficientes
¿cómo puede B. cinerea hacer frente a estas
cantidades elevadas de ROS?
Investigaciones adicionales sobre la regulación de las vías de degradación de ROS condujeron al análisis de la proteína 1 (AP-1)
activadora del factor de transcripción clave del sistema, que se ha caracterizado como un regulador de la resistencia a ROS en
células de mamíferos y hongos.

Se identificó y caracterizó la proteína activadora de Botrytis 1 homóloga (Bap1)

Un análisis funcional del factor de transcripción Bap1 (botrytis activador protein 1), que controla la expresión de la mayoría de los
genes clásicos de respuesta al estrés oxidativo (OSR) en B. cinerea (Temme y Tudzynski2009).

Los resultados confirmaron los datos obtenidos previamente para los genes clasificados como genes OSR. Todos estos
genes están directamente involucrados en la OSR y en la degradación de ROS, ya que codifican catalasas, una
glutatión reductasa, un peroxiredoxina, una glutatión peroxidasa, glutatión-S-transferasas y proteínas de la tiorredoxina así
como de los sistemas redox de glutaredoxina.

Un papel esencial del factor de transcripción Bap1 en la resistencia a ROS,

 ¿Los sistemas enzimáticos básicos de desintoxicación
de ROS son esenciales para la virulencia?

Como las ROS están presentes durante el proceso de infección de B. cinerea en sus plantas hospedantes (Tenberge 2004; von
Tiedemann 1997), se asumió que las proteínas reguladoras del equilibrio celular de ROS estaban involucradas en el proceso de
infección.

Sin embargo, en un estudio la eliminación del factor de transcripción AP-1 no condujo a una reducción de la virulencia de B. cinerea
en ninguno de los sistemas hospedantes probados .
El sistema de tiorredoxina es de gran
importancia para el mantenimiento de la
homeostasis redox celular. Aquí, mostramos
que tiene una influencia severa en la virulencia
de Botrytis cinerea,

Se sugiere que el hongo no enfrenta estrés

oxidativo en la planta, sino, que se utilizan
sistemas alternativos para asegurar la
homeostasis redox durante la infección.

EL SISTEMA DE TIORREDOXINA

El sistema de tiorredoxina está compuesto por

dos enzimas, la tiorredoxina y la tiorredoxina
reductasa. Identificamos dos genes que
codifican tiorredoxinas (bctrx1, bctrx2) y un gen
que codifica una tiorredoxina reductasa (bctrr1)
en el genoma de B. cinerea.
El sistema de tiorredoxina está compuesto por dos enzimas, la tiorredoxina y la tiorredoxina reductasa.

(Viefhues et al., 2014)

MATERIALES
Y
METODOS
 Materiales vivos utilizados

Cepa B05.10 de Botrytis cinerea es un aislado de Cepas mutantes de B05.10 con una deleción de
un tipo salvaje de Vitis vinifera algunos genes

dos genes que codifican tiorredoxinas (bctrx1, bctrx2) y

un gen que codifica una tiorredoxina reductasa (bctrr1)

Phaseolus vulgaris Tomate

Manzana
El casete génico: se refiere a un fragmento de DNA que contiene uno o más genes de interés entre uno o más puntos de restricción. Este
fragmento puede ser transferido desde una secuencia de DNA (normalmente un vector genético) a otra cortando el fragmento mediante enzimas
de restricción y ensamblándolo en la segunda secuencia.

Transformación de
hongos

Protoplastos protoplasto es una célula de plata, bacteria u hongo

que ha perdido total o parcialmente su pared
celular, para lo cual se usan mecanismos
mecánicos o enzimáticos.
Plásmido que posee el
gen de interés

Orígenes de
replicación

Procariotes
Eucariotes
 Los genes de fusión contienen marcadores de resistencia a fármacos
nat1 (nurseotricina) o hph (higromicina B).

Cassettes

El vector pCSN44. Este plásmido contiene el gen
marcador de resistencia a fármacos dominante
higromicina B fosfo transferasa (hph) de E. coli,
flanqueado por el promotor y el terminador del gen trpC
de Aspergillus nidulans.
El vector pNR1, contiene el gen marcador de
resistencia resistencia a nourseotricina (nat1) de
Streptomyces noursei y el control del promotor
oliC de Aspergillus. nidulans.
 Cepa FY834 de S. cerevisiae

Vector de clonación pRS426

Este es un vector de 2 mm que
contiene un marcador URA3 para
la selección en S. cerevisiae.

La cepa de levadura FY834 se

cotransformó con los fragmentos
amplificados, ( cassettes hph, nat1
y el plásmido con huecos pRS426)
La integración homóloga (HI) de los
casetes de resistencia en el locus
del gen de interés se demostrará
mediante PCR
 El ADN del plásmido de colonias de levadura prototróficas de uracilo se aisló utilizando el kit de aislamiento
de plásmido de levadura SpeedPrep (DualsystemsBiotech, Schlieren, Suiza) y se transformó en E. coli.
Después del aislamiento del plásmido, el casete de expresión se secuenció y extirpó usando las
enzimas ApaI y SacII y se transformó en B. cinerea

Para la transformación de B. cinerea se utilizaron 1,5 g de micelio y mezclado con una mezcla enzimática de 10 ml de
glucanex (Novozymes, Bagsværd, Dinamarca), enzima de lisis (Sigma-Aldrich, St. Louis) y Yatalase (TakaraBio Inc.,
Shiga, Japón), se incubó durante 1 hora a 28°C a 95 rpm.

Los protoplastos fueron transformado según Schulze-Gronover y asociados (2001), utilizando 20 g del vector
linealizado o 300 l de PCR
productos

Las colonias resistentes se transfirieron a placas de agar que contenían Agar GB5 y suplementado con 70 g de
higromicina B por mililitro (Invitrogen) o 70 g de nourseotricina por mililitro (Werner-Bioagents, Jena, Alemania).
Conidiosporas de heterocarióticos transformantes se recolectaron, se diluyeron con agua estéril H2O y 50 a 100
esporas sembradas en medio de selección. Después 24 h, las esporas individuales germinadas se recogieron y se
transfirieron a nuevo medio de selección. El éxito del aislamiento fue controlado por extracción de ADN genómico,
seguida de una PCR de diagnóstico y transferencias del Sur.
RESULTADOS
Identificación y caracterización de BcTrx1, BcTrx2 y BcTrr1.

motivo de tiorredoxinas : CxxC

Diagnostic PCR confirmed independent homokaryotic transformants for:

Δbctrx1 (T1, T5, T9)
Δbctrr1 (T6, T14, T15)
Δbctrx2 (T1, T3, T8)
Δbctrx1/2 (T2, T10) (data not
shown)
El sistema de tiorredoxina es crucial para la respuesta al estrés oxidativo.

Growth of Δbctrx1, Δbctrx2, Δbctrx1/2, and Δbctrr1 under oxidative stress conditions compared with wild-type B05.10. Strains were grown for 3
days on complete medium (CM) supplemented with 5, 10, or 20 mM H2O2 and 500 M menadione. A, Influence of H2O2 on fungal growth. Pictures
were taken after 3 days. B, Radial growth rates under oxidative stress conditions. Colony diameters were measured at 3 days postinoculation.
Growth of each strain on CM medium (without stressing agent) was set to 100% and relative growth is shown. Mean values and standard
deviations were calculated for three colonies per strain and condition. Three independent experiments were performed. Asterisks represent
significant differences (t-test) compared with the wild type (WT) in each condition (* = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001).
El sistema de tiorredoxina tiene un fuerte impacto en la patogenicidad.

Fig. 3. Evaluation of conidial development and differentiation of infection structures. A, Time course experiment to monitor sugar-
induced conidial germination. Conidia were prepared in GB5 + 10 mM glucose and dropped on a glass slide. Scale bar = 20 m. B,
Germination rates of Δbctrx1, Δbctrr1, and wild type (WT) conidia in GB5 + 10 mM glucose on a glass surface. Represented are the
mean values and standard deviations of two independent experiments with 100 conidia per strain.
El sistema de tiorredoxina tiene un fuerte impacto en la patogenicidad.

C, Visualization of infection structures. Onion epidermis was inoculated with mycelia (left) and conidia (right) and was incubated for
24 h. Lactophenol blue was used to stain conidia and hyphae on the surface, while invading hyphae remain colorless. Scale bar = 20
m. D, Scanning electron microscopy (SEM) images of Botrytis cinerea–infected bean leaves. Conidia suspension (2 × 106 spores
per milliliter) of Δbctrx1, Δbctrr1, and WT were dropped on detached bean leaves and were incubated for 18 h. The samples were
prepared for SEM by glutaraldehyde fixation, ethanol series, critical point drying, and gold sputter-coating. Arrows indicate
appressoria-like structures. Scale bars = 15 m.
Fig. 4. Pathogenicity assays with mutants of the thioredoxin system. A, Infection of primary bean leaves of Phaseolis vulgaris. Leaves were
inoculated with droplets of conidial suspension and were incubated for 7 days. B, Infection of detached tomato fruits and apples with droplets
of conidial suspension of Δbctrx1 and Δbctrr1 in comparison with the wild type (WT). Apples were cut and tomatoes were wounded prior to
infection. The infection was monitored for 7 days. Pictures were taken at 3 and 4 days postinoculation (dpi), respectively. C, Measurement of
lesion diameter of indicated mutants after inoculation of wounded and unwounded bean leaves. Shown are the mean diameters and standard
deviations of two independent experiments with 24 infections per strain and condition. Asterisks represent significant differences compared
with the WT in each condition (* = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001).
D, Plant infection with nonsporulating mycelia and measurement of resulting lesion diameters at 2 dpi. Shown
are the mean values and respective standard deviations. In two independent experiments, 24 infections per
strain were analyzed. Asterisks represent significant differences (t-test) compared with the WT in each
condition (* = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001).
Fig. 5. Reactive oxygen species production and influence of antioxidants. A,
Diaminobenzidine (DAB) test for the analysis of H2O2 production of indicated
mutants compared with the wild type. Fresh mycelium was weighed and 1 ml of
DAB solution was added. Incubation took place for 1.5 h in the dark. NC = negative
control, DAB solution + 1 l H2O2; PC = positive control, DAB solution + 1 l H2O2 +
1 l horseradish peroxidase. Three independent experiments in triplicate per strain
were performed. B, DAB assay on solid medium. The strains were grown for 2 days
on complete medium (CM) or CM + 750 M dithiothreitol (DTT). The plates were
flooded with DAB solution and were incubated for 2 h in darkness. Images were
taken the following day. C, Superoxide detection of Δbctrx1, Δbctrr1, and WT via
nitro blue tetrazolium (NBT); 0.05% (wt/vol) NBT aqueous was added to the
conidial suspensions (1 × 105 spores per milliliter). As a control, 50 M
diphenyleneiodonium (DPI) was added prior to supplementation of NBT. The
staining was monitored by light microscopy. D, In planta infection assay with
ascorbic acid of Δbctrx1, Δbctrr1, Δbcltf1, and the WT. Conidia suspensions (2 ×
105 spores per milliliter) were supplemented with 5 g of ascorbic acid per liter prior
to inoculation. The pictures were taken at 3 days postinoculation. E, Growth assay
of Δbctrr1, Δbcltf1, and WT under addition of sorbitol, DTT, and ascorbic acid,
respectively. CM was supplemented with 1 M sorbitol, 750 M DTT, and 5 g of
ascorbic acid per liter. The plates were incubated for 3 days under light and dark
conditions. Given are the mean values and standard deviations resulting from two
independent experiments with five plates per strain and condition. Asterisks
indicate significant differences (t-test) compared with the growth on CM for each
condition and strain (* = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001).

Δbcltf, un mutante que carece de

un factor de transcripción GATA
sensible a la luz
 Fig. 6. Effect of bctrr1 and bctrx1 deletion on
gene expression of general and oxidative
stress genes. Northern blot analysis of stress
response gene expression. Strains were
cultivated in liquid Czapek Dox minimal
medium supplemented with 10 mM H2O2 (+)
or without H2O2 (–) for 30 min.

The OSR (oxidative-stress-resistance)

Fig. 7. Cellular localization of BcTrx1, BcTrr1, and BcTrx2 and
analysis of the glutathione redox status. A, Overnight-
germinated hyphae of bctrx1-gfp and bctrr1-gfp were analyzed
via epifluorescence microscopy (left: green fluorescent [GFP],
right: bright field). Scale bar = 10 m. B and C, BcTrx2
localization by microscopic analysis of bctrx2-gfp germinating
hyphae. For co-staining experiments the ER-Tracker Blue-
White DPX or Hoechst dye was used. B, From left to right:
GFP, ER-tracker, bright field. C, GFP (top), Hoechst (bottom).
Scale bar = 10 m.
Fig. 8. Virulence and growth assay as well as evaluation of germination and penetration abilities of Δbcglr1 and Δbcglr2. A, Infection
of primary bean leaves of Phaseolis vulgaris with conidia suspension of Δbcglr1 and Δbcglr2 in comparison with the wild type (WT).
Progression of infection was documented at 2, 4, and 7 days postinoculation (dpi). B, Examination of penetration (top) and
germination (bottom) of Δbcglr1, Δbcglr2, and WT spores after 24 h. For analysis of infection structures, onion epidermal strips were
inoculated with conidia suspension (1 × 105 spores per milliter and H2O); for germination, conidia (1 ×105 spores per milliliter and
GB5 + 10 mM glucose) were dropped on glass slides and allowed to germinate overnight. Scale bar = 20 m.
 C, Growth assay of Δbcglr1 and Δbcglr2 on Czapek Dox minimal medium (CD) in comparison with
WT. In addition, CD was supplemented with 2 mM cysteine and methionine and growth was
measured at 2 dpi. The growth rate on CD was set to 100% and growth on supplemented media
was calculated in relation to the standard medium. The experiments were performed three times
with three replicates per strain and condition. Significant differences (t-test) of mean values are
represented by asterisks (* = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001).
 OTROS
EJEMPLOS
(Fan et al., 2019)
FIGURE 4 | Pathogenicity and TRI expression assays of
the FgTRR deletion mutants. (A) Flowering wheat heads
were inoculated with conidial suspensions and examined
14 days post-inoculation (dpi). (B) Corn silks were
inoculated with mycelial plugs and examined 5 dpi. Bar =
5 mm. (C) Disease index was examined from the number
of symptomatic spikelets per wheat head 14 dpi.
Different letters indicate statistically significant difference
(P < 0.05) by Duncan’s multiple range test. (D) Relative
expression levels of TRI5, TRI6, and TRI10 in PH-1 and
the 1TRR#1 mutant. The GAPDH gene was used as an
internal control. Asterisks represent statistically
significant difference (P < 0.05) by Student’s t-tests
 The histological examination of hyphal growth and inhibition rate between Sclerotinia sclerotiorum
strain 1980 and SsTrx1-01, SsTrx1-02, and SsTrx1-03 transformants in potato dextrose agar (PDA)
supplemented with different H2O2 concentrations. (a) Nitroblue tetrazolium stained hyphae at 2
days postinoculation and hyphal growth at 0, 5, 10, and 15 mM H2O2 between S. sclerotiorum
(Yijuan Kusom, 2019) strain 1980 and SsTrx1-01 at 72 hr postinoculation (hpi). (b) The inhibition rate in PDA with 5, 10,
and 15 mM H2O2 concentrations at 12, 24, 36, 48, 60, and 72 hpi. The bars indicate the standard
error and asterisks (**) denote significant differences (p < .01, one-way analysis of variance). (c)
Relative gene expression of SsTrx1 in hyphae treated with 10 mM H2O2 as compared with 5 mM
H2O2 and control
 Effect of T-2 on the contents of flavonoids, total phenolics in grape berries. (a) Flavonoids
content in grape berries. The horizontal axis is the storage days after inoculation, the
(Wu et al., 2022) vertical axis is the contents of flavonoids (mg/g). (b) Total phenolics content in grape
berries. Samples were measured separately at 1 to 5 DAI. The horizontal axis is the
storage days after inoculation, the vertical axis is the contents of total phenolics (mg/g).
The error line is the standard deviation of multiple biological replicates; results are
mean ± standard deviation and the analysis of the significance of the data differences was
performed using the t-test; *p < .05; **p < .01
Effect of T-2 on antioxidant enzymes in grape berries. (a) Changes in peroxidase (POD) activity. The horizontal axis is the
storage days after inoculation, the vertical axis is activity of POD (U/g FW). (b) Changes in catalase (CAT) activity. The
horizontal axis is the storage days after inoculation, the vertical axis is the activity of CAT (U/g FW). (c) Changes in phenylalnine
ammonia-lyase (PAL) activity. The horizontal axis is the storage days after inoculation, the vertical axis is activity of PAL (U/g
FW) samples that were measured separately at 1 to 5 DAI. The error line is the standard deviation of multiple biological
replicates; results are mean ± standard deviation and the analysis of the significance of the data differences was performed using
the t-test; *p < .05; **p < .01
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GRACIAS