Grapevine (Vitis vinifera) is one of the most economically important fruit crops worldwide, but many cultivars conventionally used for table grape and wine production are susceptible to two several diseases, such as downy mildew (caused by Plasmopara viticola) and powdery mildew (caused by Erysiphe necator). These pathogens are controlled by frequent applications of chemical fungicides to avoid significant losses in yield and berry quality. However, the ecological drawbacks of pesticides and the rapid appearance of resistant pathogen strains have sparked crescent interest in sustainable control alternatives. The development of resistant grapevine hybrids represents a promising option to limit the application of chemical pesticides in viticulture. Thus, a better understanding of the mechanisms responsible for the grapevine defense against downy and powdery mildew and the identification of key resistance genes will help breeding programs to develop resistant varieties. In addition to the resistance genes, the susceptibility genes are key regulators of defense processes in plants, such as the Mildew Locus O (MLO) genes that are known to be responsible for powdery mildew susceptibility in herbaceous plants. Gene expression profiling has been used extensively to investigate regulation processes of the grapevine interaction with mildews and to identify gene markers of the grapevine resistance. However, all studies to date have involved the use of whole leaves, while only a small fraction of host cells is in contact with the pathogen at the early infection stages. Therefore, highly localized transcriptional changes of infected cells may be masked by the large portion of non-infected cells when analyzing the whole leaf. The final goal of this PhD project was to develop innovative methods for disease control, such as resistant varieties and efficient resistance inducers, based on a better understanding of the key genes and cellular processes involved in the defense and susceptibility mechanisms of the grapevine against downy and powdery mildew. Specific aims were i) to characterize the grapevine response in the sites of downy mildew infection in order to identify site-specific transcriptional regulations in infected stomata, in surrounding areas and in distal part non-infected by the pathogen, and ii) to silence four grapevine MLO genes (MLO-6, MLO-7, MLO-11 and MLO-13) through RNA interference in order to identify the key genes responsible for powdery mildew susceptibility. For the characterization of the site-specific transcriptional regulations against downy mildew, a laser microdissection protocol was optimized to collect stomata and surrounding cells from leaves of in vitro-grown grapevines at early stages of pathogen infection, and the expression levels of ten genes involved in the grapevine response to downy mildew were investigated by real time PCR. The expression levels of seven P. viticola-responsive genes were greater in microdissected cells than in whole leaves, highlighting the site-specific transcriptional regulation of the host response. The gene modulation was restricted to the stomata cells and to the surrounding areas of infected tissues, indicating that short-distance signals are implicated. The high sensitivity of the laser microdissection analysis showed significant modulations of three genes that were completely masked in the whole tissue analysis. For the characterization of susceptibility genes, three grapevine lines silenced in the expression of MLO genes showed an increased resistance against powdery mildew. Expression analyses of resistant and susceptible lines demonstrated the key role of the MLO-7 gene in the susceptibility of powdery mildew, with an additive contribution of the MLO-6 and MLO-11 genes. The expression level of thirteen defense-related genes was lower in resistant than in susceptible lines, highlighting an early MLO-dependent reduction of powdery mildew invasion. However, the accumulation of defense-related compounds was comparable in resistance and susceptible lines, suggesting that further analysis are required to better clarify the complex resistance mechanisms against powdery mildew. The protocol for the laser microdissection analysis optimized in this study could greatly increase the sensitivity of further transcriptomic studies to identify new marker genes of the grapevine defense activated at the sites of infection. Specifically, the three MLO genes identified in this project represent novel markers of grapevine resistance and they could be used for marker-assisted selection in breeding programs of powdery mildew-resistant grapevines.

Lenzi, Luisa (2016-05-05). Characterization of mechanisms of grapevine response to downy and powdery mildew through laser microdissection and gene silencing. (Doctoral Thesis). Università degli Studi della Tuscia, Viterbo, a.y. 2015-2016, Corso di dottorato di ricerca in Genetica e Biologia Cellulare, Ciclo XXVIII, FIRST. handle: http://hdl.handle.net/10449/34030

Characterization of mechanisms of grapevine response to downy and powdery mildew through laser microdissection and gene silencing

Lenzi, Luisa
2016-05-05

Abstract

Grapevine (Vitis vinifera) is one of the most economically important fruit crops worldwide, but many cultivars conventionally used for table grape and wine production are susceptible to two several diseases, such as downy mildew (caused by Plasmopara viticola) and powdery mildew (caused by Erysiphe necator). These pathogens are controlled by frequent applications of chemical fungicides to avoid significant losses in yield and berry quality. However, the ecological drawbacks of pesticides and the rapid appearance of resistant pathogen strains have sparked crescent interest in sustainable control alternatives. The development of resistant grapevine hybrids represents a promising option to limit the application of chemical pesticides in viticulture. Thus, a better understanding of the mechanisms responsible for the grapevine defense against downy and powdery mildew and the identification of key resistance genes will help breeding programs to develop resistant varieties. In addition to the resistance genes, the susceptibility genes are key regulators of defense processes in plants, such as the Mildew Locus O (MLO) genes that are known to be responsible for powdery mildew susceptibility in herbaceous plants. Gene expression profiling has been used extensively to investigate regulation processes of the grapevine interaction with mildews and to identify gene markers of the grapevine resistance. However, all studies to date have involved the use of whole leaves, while only a small fraction of host cells is in contact with the pathogen at the early infection stages. Therefore, highly localized transcriptional changes of infected cells may be masked by the large portion of non-infected cells when analyzing the whole leaf. The final goal of this PhD project was to develop innovative methods for disease control, such as resistant varieties and efficient resistance inducers, based on a better understanding of the key genes and cellular processes involved in the defense and susceptibility mechanisms of the grapevine against downy and powdery mildew. Specific aims were i) to characterize the grapevine response in the sites of downy mildew infection in order to identify site-specific transcriptional regulations in infected stomata, in surrounding areas and in distal part non-infected by the pathogen, and ii) to silence four grapevine MLO genes (MLO-6, MLO-7, MLO-11 and MLO-13) through RNA interference in order to identify the key genes responsible for powdery mildew susceptibility. For the characterization of the site-specific transcriptional regulations against downy mildew, a laser microdissection protocol was optimized to collect stomata and surrounding cells from leaves of in vitro-grown grapevines at early stages of pathogen infection, and the expression levels of ten genes involved in the grapevine response to downy mildew were investigated by real time PCR. The expression levels of seven P. viticola-responsive genes were greater in microdissected cells than in whole leaves, highlighting the site-specific transcriptional regulation of the host response. The gene modulation was restricted to the stomata cells and to the surrounding areas of infected tissues, indicating that short-distance signals are implicated. The high sensitivity of the laser microdissection analysis showed significant modulations of three genes that were completely masked in the whole tissue analysis. For the characterization of susceptibility genes, three grapevine lines silenced in the expression of MLO genes showed an increased resistance against powdery mildew. Expression analyses of resistant and susceptible lines demonstrated the key role of the MLO-7 gene in the susceptibility of powdery mildew, with an additive contribution of the MLO-6 and MLO-11 genes. The expression level of thirteen defense-related genes was lower in resistant than in susceptible lines, highlighting an early MLO-dependent reduction of powdery mildew invasion. However, the accumulation of defense-related compounds was comparable in resistance and susceptible lines, suggesting that further analysis are required to better clarify the complex resistance mechanisms against powdery mildew. The protocol for the laser microdissection analysis optimized in this study could greatly increase the sensitivity of further transcriptomic studies to identify new marker genes of the grapevine defense activated at the sites of infection. Specifically, the three MLO genes identified in this project represent novel markers of grapevine resistance and they could be used for marker-assisted selection in breeding programs of powdery mildew-resistant grapevines.
Perazzolli, Michele
Grapevine
Downy mildew
Powdery mildew
Mlo genes
Gene expression
Stomata
Laser microdissection
RNA interference
Settore BIO/10 - BIOCHIMICA
2015-2016
Corso di dottorato di ricerca in Genetica e Biologia Cellulare, Ciclo XXVIII
FIRST
Lenzi, Luisa (2016-05-05). Characterization of mechanisms of grapevine response to downy and powdery mildew through laser microdissection and gene silencing. (Doctoral Thesis). Università degli Studi della Tuscia, Viterbo, a.y. 2015-2016, Corso di dottorato di ricerca in Genetica e Biologia Cellulare, Ciclo XXVIII, FIRST. handle: http://hdl.handle.net/10449/34030
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