| Botrytis cinerea belongs to necrotrophic fungi and causes grey mold disease on wide ranges of crop plants. Most of the crop plants lack effective resistance against B. cinerea; however, plants have developed a variety of defense mechanisms to protect themselves against B. cinerea infection. In this study, we adapted virus-induced gene silencing (VIGS) technology to explore the possible functions of histone H2B E3ubiquitin ligases SlHUB1and SlHUB2, mediator complex subunits and the de novo biosynthesis of vitamin B6pathway in tomato resistance response to B. cinerea.Histone monoubiquitination formed ub-H2B mainly binds to chromatin to activate gene transcription and mRNA elongation.In Arabidopsis, histone H2B mono-ubiquitination pathway is not only involved in growth and development, but also has a regulatory role in defense responses. However, the function of histone ubiquitination pathway in tomato disease resistance is still not clear yet. In this study, we identified two genes encoding homologous proteins of Arabidopsis HUB1and HUB2, called SlHUB1and SlHUB2, and in vitro experiments demonstrated that the cloned tomato SlHUB1and SlHUB2had H2B monoubiquitination E3ligases activity. Silencing of either SlHUBl or SIHUB2in tomato plants resulted in significant reduction of resistance to B. cinerea when compared to control plants. Previous study has shown that the Arabidopsis HUB1interacts with MED21, which is involved in resistance against B. cinerea. By contrast, although tomato SlHUB1interacted with SlMED21in yeast, silencing of SIMED21did not affect the resistance to B. cinerea, indicating a possibility that S1MED21is not involved in the function of the SlHUB1-regulated resistance to B. cinerea in tomato. Expression analysis showed that SlHUB1and SIHUB2can be induced by B. cinerea, SA or ACC. Further studies have shown that, SlHUB1-and SlHUB2-silenced plants had reduced cell wall thickness, and increased leaves water loss rates, as compared to the control plants. After B. cinerea infection, SlHUB1-and SlHUB2-silenced plants accumulated more reactive oxygen and callose compared to the control plants. qRT-PCR results revealed that, in SIHUB1-and SlHUB2-silenced plants, the expression of genes involved in lignin synthesis was decreased, SA pathway genes were significantly upregulated, whereas JA/ET pathways genes expression was significantly decreased, as comparied to those in the control plants. Taken together, our data demonstrate that S1HUB1and S1HUB2play an important role in tomato resistance to B. cinerea, probably through regulation of cell wall formation and the JA/ET signaling pathway.Mediator complex is a multi-functional protein complex conserved in eukarytes, and plays important roles in the transcriptional mechanism as RNA polymerase II cofactor. In this study, we identified25genes(SIMED) that encode mediator complex subunits through bioinformatics approach using Arabidopsis mediator complex subunits as queries. qRT-PCR analysis of SIMEDs expression revealed that when compared to those in the mock-inoculated plants, the expression levels of SIMED6, SIMED12, SIMED13, SIMED14, SIMED15, SlMED19a, SIMED23, SIMED25, SIMED31, SIMED36and SIMED37a in B. cinerea-inoculated plants increased more than3-fold; the expression levels of SlMED7a, SIMED13, SlMED28, SIMED31and SIMED34increased more than3-fold by SA; the expression levels of SlMED5a, SIMED12, SIMED13, SIMED14, SIMED16and SIMED25showed3-fold increase by ACC. VIGS-based functional analysis of individual SIMED gene revealed that the SIMED8-, SIMED10b-, SIMED12-, SIMED14-, SIMED15-, SIMED16-, SIMED17-or SIMED25-silenced plants showed growth defects, such as slowly growth rate or leaf malformations, indicating that these SIMED genes have functions in tomato growth and development. We found that silencing of SlMED5b, SIMED8, SIMED15, SIMED16, SIMED17, SIMED18, SlMED20b, SIMED25or SIMED28affected the B. cinerea resistance in tomato. The functions of SIMED8, SIMED15, SlMED16, SIMED18and SIMED25in B. cinerea resistance are similar to those in Arabidopsis; however, we idenrified4novel sbunits, SlMED5b, SIMED17, SlMED20b and SlMED28that are involed in disease resistance against B. cinerea. Our data indicate that SIMED17and SlMED20b may positively regulate tomato resistance to B. cinerea, whereas SlMED5b and SlMED28may negatively regulate tomato resistance to B. cinerea.Vitamin B6(VB6), an essential cofactor for numerous metabolic enzymes, has recently been shown to act as a potent antioxidant and play important roles in developmental processes and stress responses. However, little is known about the potential function of VB6in plant disease resistance response against pathogen infection.In the present study, we explored the possible involvement of VB6in defense response against B. cinerea through functional analysis of tomato VB6biosynthetic genes. Three de novo VB6biosynthetic genes, SIPDX1.2, SlPDX1.3and SIPDX2, and one salvage pathway gene SISOS4were identified and the SIPDX1.2, SlPDX1.3and SIPDX2genes were shown to encode functional enzymes involved in de novo of biosynthesis VB6, as revealed by complementation of the VB6prototrophy in yeast snzl and snol mutants. Expression of SIPDX1.2, S1PDX1.3and SISOS4genes was induced by infection with B. cinerea. VIGS-mediated knockdown of SIPDX1.2or SlPDX1.3but not SIPDX2and SISOS4led to increased severity of disease caused by B. cinerea, indicating that the VB6de novo biosynthetic pathway but not the salvage pathway is involved in tomato defense response against B. cinerea. Furthermore, the SIPDX1.2-and SIPDX1.3-silenced tomato plants exhibited reduced levels of VB6contents and reactive oxygen species scavenging capability, increased levels of superoxide anion and H2O2generation, and increased activity of superoxide dismutase after infection by B. cinerea. Our results suggest that VB6and its de novo biosynthetic pathway play important roles in regulation of defense response against B. cinerea through modulating cellular antioxidant capacity. |
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