Researches On The Mechanism For Chineses Wild Vitis Germplasm Leaves Against Botrytis Cinerea

Grapes, one of the most importantly economic fruits are represented by Vitis vinifera widely cultivated in the world but susceptible to pathogens including necrotrophic fungus Botrytis cinerea. The disease caused by B. cinerea is threating grape industry in our county and results in huge economic damage, especially with the development of the protected cultivation. The disease management is still largely depended on unsustainable chemical control. Therefore, the researches on long-term and effective resistance breeding germplasm and new resistant grapes towards B. cinerea have been focused on. However, the resistant grapes to B. cinerea is rarely revealed, especially for the rich Chinese wild Vitis germplasm which have been confirmed with high resistance to many pathogens. Moreover, the resistance characteristics and potential mechanism of grapes against B. cinerea is poorly understand. In this study, we proceeded the leaf resistance evaluation of Chinese wild Vitis genotypes and selected highly resistant wild Vitis and susceptible cultivars to comparatively analysis the differences in B. cinerea development, ROS(Reactive Oxygen Species) and antioxidative enzyme responses in the interactions with B. cinerea. Furthermore, differently expression genes and responsible biology functions involved in two grapes of different leaf resistance levels at different infection stages by B. cinerea were also detailedly analyzed through the large scale sequencing technology of RNA-Seq. This study supplied some reference basis for breeding new grape germplasm with good resistance to B. cinerea. The main results were as follows:1. A screen of 41 Vitisgenotypes for leaf resistance to B. cinerea, including 30 genotypes from Chinese wild Vitiswere proceeded and the results proved thatmany Chinese wild Vitis genotypes were resistant. However most of the investigated V. vinifera, or its hybrids, were susceptible. The six most highly resistant genotypes were all belonged to Chinese wild Vitis species: V. sp.(Qinling grape)‘Pingli-5', V. yenshanensis‘Yanshan-1', V. adstricta Hance‘Taishan-2' and V.amurensis Rupr‘Shuangyou', ‘Tonghua NO.3' and ‘Taishan-11'.2. B. cinereawas severely blocked on leaves of the particularly resistant Chinese wild V. sp.(Qinling grape)‘Pingli-5' and V.amurensis Rupr‘Shuangyou' while built a successful necrotrophic interaction with the very susceptible V. vinifera cv ‘Red Globe'. ‘Pingli-5' of high resistance produced very little ROS and timely evaluated the antioxidative activities in response to B. cinerea infection. However ‘Red Globe' suffered the colonization of B. cinerea and huge oxidative stress, which was associated with its much lower antioxidative ability than that triggered in ‘Pingli-5'.3. RNA-Seq transcriptomic profiles after six different stages induced by B. cinerea of leaves of the highly resistant Chinese wild V.amurensis ‘Shuangyou' and the susceptible V. vinifera cv ‘Red Globe' were analyzed in details. Comparatively analysis revealed 7289 differently expression genes(DEGs) totally and lager transcriptional change in ‘Red Globe' than in ‘Shuangyou', except for 8 hpi(hours post infection) with more DEGs in ‘Shuangyou'. We found different changes of biology process in multiple levels during the both interactions GO, KEGG pathway and gene expression clusters.4. JA/ETH and ABA etc. signal pathways were all induced in both resistant Chinese wild V.amurensis‘Shuangyou' leaves and susceptible V. vinifera cv ‘Red Globe' leaves in response to B. cinerea, yet with a lower level in ‘Shuangyou'. Genes involved in both positively and negatively regulating signal pathways performed higher expression levels in ‘Red Globe' than ‘Shuangyou'. It might exist that higher level of signal pathways simultaneously induced in ‘Red Globe' could not promote effective defenses. Contrarily, lower levels in ‘Shuangyou' were suggested to be moderate for effective defenses.5. Degrading and reinforcing cell wall were both significantly induced in the two grape leaves after the infection of B. cinerea. However, responses on the surface structure were different at 8 hpi, which was indicated by the only decrease of epidermal cuticle and cell wall biosynthesis in Chinese wild V.amurensis‘Shuangyou' leaves 8 hpi. Until 36 hpi, it decreased more in susceptible V. vinifera cv ‘Red Globe' than ‘Shuangyou'. The responses at 8 hpi in ‘Shuangyou' might lead itself speed the recognition and defense response with rapid excretion of phytoalexins(especially triterpenoids) to resist B. cinerea through increasing the permeability of its leaf surface structure.6. B. cinerea triggered defense responses of secondary metabolism both in leaves of resistant Chinese wild V.amurensis‘Shuangyou' and in susceptible susceptible V. vinifera cv ‘Red Globe', yet, much higher in the latter, which indicated the requirement for more materials and energy. Photosynthesis was declined more but glycolysis and TCA cycle were elevated higher in ‘Red Globe' than in ‘Shuangyou'. It was found that both ‘Shuangyou' and ‘Red Globe' might increase the GABA shunt from glutamate(Glu) to complement energy. However, the interaction with ‘Red Globe' might lead excessive consumption, especially for Glu metabolism without continuous Glu supply by Glu generation associated genes. Comparatively, ‘Shuangyou' regulated multiple metabolism processes just in time and quantity, especially with higher continuously expression of Glu generation associated genes. This might supply ‘Shuangyou' enough energy for defenses and also balance its metabolism so as to achieve the resistance.7. The higher expression levels of respiratory burst homology 1, GST, Nudix hydroxylase, ABC transporters and MATE in leaves of susceptible V. vinifera cv ‘Red Globe' than resistant Chinese wild V.amurensis‘Shuangyou' reflected that the former suffered much severe oxidative stress, more toxic accumulation and imbalance of cellular homeostasis. Biological rhythm process in ‘Red Globe' was down-regulated at 12 hpi, which would cause cellular turbulence and death, but this did not occurred in ‘Shuangyou'. Moreover, Nudix hydroxylase, NAC, cysteine-rich recepter-like kinase, and cysteine-rich secretory protein playing a role in senescence and cell death accompanied with Mlo genes involved in inhibiting cell death were all much highly induced in ‘Red Globe' than those in ‘Shuangyou'. All these results suggested that ‘Red Globe' infected byB. cinerea had to face more pressure of imbalance of cellular homeostasis and cell senescence and cell death.8. Genes that performed higher basic expression levels in leaves resistant Chinese wild V.amurensis‘Shuangyou' of than in susceptible V. vinifera cv ‘Red Globe' could endow ‘Shuangyou' much stronger innate immunity, which indicated that it can innate the defense against B. cinerea rapidly at the first time of the interaction. These defense responses could be associated with directly structure resistance and energy supply, higher ability to keep cellular homeostasis, DNA repair and suppress cell death and more fast recognition and signaling. However, ‘Red Globe' might have to proceed for a longer time to innate defenses responses to B. cinerea. Thus, a direct and rapid basic defense and a later defense might be one of the important reasons, respectively for ‘Shuangyou' resistant and ‘Red Globe' susceptible to B. cinerea.