| Late blight (LB), caused by oomycetes pathogen Phytophthora infestans, is considered as a major threat to tomato and potato production worldwide. To combat with this destructive disease, the interaction between the Solanaceous family and this pathogen has been well documented in the past decades. Age-related resistance (ARR) existed in many plant-pathogen interactions was also found in Solanaceae when response to this notorious pathogen. However, little information has been obtained on the important vegetable crop tomato. In the present search, characterization of physiological races with some collected isolates and evaluation of wild tomato species Solanum pimpinellifolium with a global collection against P. infestans were conducted. ARR involved in resistance (R) gene and quantitative trait loci (QTL) were analyzed, role of phytohormone pathways (salicylic acid, ethylene and jasmonic acid) mediated Ph-3resistant gene were investigated. The major results obtained are listed as the following:1. By using different tomato plants comprising Ph-1, Ph-2, and Ph-3gene, eight isolates of P. infestans collected from Fujian province, the south part of China, were characterized. Results indicated that the most virulent race is T1,2among all tested isolates. Further, in total418S. pimpinellifolium accessions collected globally were screened for the resistance to P. infestans race T1,2,4. With this wide range of collections,19accessions have been identified to give different levels of resistances. Among these resistant accessions, three accessions include LA2001, LA2147, LA3078were identified as highly resistant and two accessions LA1575and LA1634were identified as moderate resistant. All of these accessions were not identified previously. So far, all identified R genes conferring resistance to P. infestans in tomato are derived from S. pimpinellifolium. The geographical distribution and genetic variation within these identified resistant accessions were analyzed by using the collection site data and genome sequence information. We demonstrated that most of the resistant accessions were from Peru. In total,19accessions were divided into four groups by using18polymorphic SNP markers and most of them (15accessions), including the most effective resistant Ph-3gene, were grouped to one large cluster. To decipher the co-evolution between S. pimpinellifolium and P. infestans, detailed information about these R genes and pathogen is in progress.2. Results derived from disease tests on six tomato genotypes, including Solanum lycopersicum CLN2037B and S. pimpinellifolium L3708previously proved to harbor Ph-3gene, and three S. habrochaites LA2099, LA1033and LA1777previously proved to possess QTLs conferring resistance to late blight, along with S. lycopersicum LA2818as susceptible control, demonstrated that both qualitative and quantitative resistance presented a significant ARR effects to P. infestans along with the increased plant age. The susceptible control plants showed the same behavior but with less pronounced increase. Hence common defense mechanism in tomato against P. infestans through ARR should be existed. To further monitor the infection course involved in ARR, both resistant genotype with Ph-3gene and susceptible genotype were investigated at the different age plants. The results indicated that the zoospores of P. infestans start to germinate within6hours post inoculation (hpi) in both genotypes. However, the resistant genotype started to show hypersensitive response (HR) at24hpi in both30and60-days old plants, and extensive cell death was observed at48hpi indicating a retard disease progress. In conclusion the developed age plays a pivotal role in Ph-3gene mediated ARR.3. In order to study the roles of phytohormone signaling pathways in the Ph-3based resistance, three complementary strategies were used. First of all, the expressions of marker genes PR-2, Chitinase9, and LOXD for SA, ET, and JA, respectively, are quantified by quantitative RT-PCR. Results showed that SA pathway induction started at24hpi in the resistant genotype with continuous increase of expression at96hpi. The up regulations of ET pathway were observed at96hpi in plants harboring Ph-3gene. It suggests that the synergism between SA and ET pathway at late time points was involved in the defense mechanism in Ph-3based resistance against P. infestans. Whereas, expression level in JA pathway was not changed at late time-points, indicating that this pathway is not involved in Ph-3based resistance against late blight. Second, In order to confirm the role of signaling pathways involved in the Ph-3based resistance, we used four tomato mutants namely Nr (ET-insensitive), epi (ET-overexpressor), def-I (JA-deficient), and NahG (line overexpressing the salicylate hydroxylase gene), and crossed them with Ph-3genotype. Plants harboring homozygote Ph-3allele were selected from the F2population and used to evaluate the responses to LB infection. For testing the involvement of SA and ET pathways, F, plants derived from cross between resistant Ph-3plants with NahG or Nr mutants, respectively, were tested. We observed restored susceptibly of individuals in case of ET pathway mutants Nr and Epi, as well as SA deficient mutants NahG, whereas, JA pathway mutants Def-1did not show any susceptible plant in the tested F2population. Finally, in order to confirm the positive role of the ET pathway, virus-induced gene silencing (VIGS) was applied to silence CTR1gene in Ph-3genotype. Results showed that silencing CTR1has positive impact on resistance conferred by Ph-3gene. Consequently, these results indicate that SA and ET pathways has positive role and actively involved in Ph-3based resistance in tomato. |
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