Association Between The Expression Of Receptor Kinase Gene LRR-RK4 And Resistance To Tobacco Red Spot Disease And Its Response To Nitrogen Application Rate

Tobacco brown spot disease is a leaf spot disease caused by Alternaria alternaria.It mainly occurs in the mature stage of tobacco leaves,has the characteristics of short gleying period and rapid outbreak,and seriously affects the production and quality of tobacco leaves,and brings immeasurable losses to tobacco farmers.Field production practice found that there were significant differences in the incidence of tobacco brown spot disease under different nitrogen levels,and the incidence of brown spot disease was also different among different tobacco varieties.LRR-RKs were long been demonstrated to be the largest subfamily of transmembrane-like receptor kinases in plants that regulate plant development and multiple defense-related responses,including cell proliferation,stem cell maintenance,hormonal signaling,host-specific and non-host-specific defense responses.However,it is currently unknown whether LRR-RKs regulate Nicotiana attenuata resistance to A.alternata,a notorious fungal pathogen causing tobacco brown spot disease.During transcriptome analysis,we identified a highly induced receptor kinase(NaLRR-RK4)in N.attenuata leaves after A.alternata inoculation.Based on this,N.attenuata and N.tabaccum(K326 and HD)were used as experimental materials.To study the expression of receptor kinase LRR-RK4 in tobacco infected by A.alternata in wild and cultivated tobacco and the molecular mechanism of its relationship with the occurrence of brown spot disease,and to explore its response to nitrogen fertilizer application,so as to provide a theoretical basis for the green prevention and control of brown spot disease in production.The main results are as follows:1,NaLRR-RK4 / Nt LRR-RK4 is a key response gene to the infection of A.alternaria in tobacco.Transcriptome experimental data found heavily induced the receptor kinase gene NaLRR-RK4 in wild tobacco after infection with A.alternaria.VIGS and RNAi silences showed that the necrotic plaques of NaLRR-RK4-RNAi and VIGSNaLRR-RK4 plants were significantly larger than those of wild control plants after infection by A.alternaria.The necrotic plaques of Nt LRR-RK4-RNAi plants of cultivated tobacco K326 and HD were significantly larger than those of control plants after A.alternaria infection.NaLRR-RK4/Nt LRR-RK4 was identified as a key response gene against A.alternaria infestation in tobacco.2,NaLRR-RK4 is involved in the resistance to tobacco brown spot disease by regulating NaERF109,defensin and Scopoletin.Transcriptomic sequencing was performed after inoculation with A.alternaria in both NaLRR-RK4-RNAi silenced plants and wild plants.The results showed that the expression levels of transcription factor gene NaERF109,defensin gene NaDEF19 and NaF6’H1,a key gene for plant protection in NaLRR-RK4-RNAi silenced plants were decreased compared with the control.Expression levels of NaDEF19 and NaF6’H1 decreased in NaERF109 plants from VIGS.These results indicated that NaLRR-RK4 positively regulated the transcription factor NaERF109,and NaERF109 regulated NaF6’H1 and NaDEF19 and participated in tobacco disease resistance.3,Different nitrogen application rates had important effects on the occurrence of brown spot disease in different cultivated tobacco varieties,the incidence of HD was more serious than that of K326 at the same nitrogen level.Cultivated tobacco K326 and HD were treated with different nitrogen application rates,and their disease conditions were investigated after inoculation with pathogens.The statistical experiment of disease index showed that the degree of disease occurrence had a certain rule with varieties and nitrogen application amount: with the extension of time after tobacco inoculation,the disease gradually worsened.Compared with different nitrogen application rates,the incidence of brown spot disease generally increased with the increase of nitrogen application rate,and at the same time point,under the same nitrogen level,the incidence of HD was more serious than that of K326.4,Nitrogen fertilizer inhibited Nt LRR-RK4,Nt ERF109,Nt F6’H1and Nt DEF19 expression in cultivated tobacco,and their expression was significantly higher in K326 plants at the same nitrogen level than in HD.Different doses of nitrogen fertilizer were applied to cultivated tobacco(K326 and HD),and samples were detected after inoculation with A.alternaria.The results showed the expression level of Nt LRR-RK4,Nt ERF109,Nt F6’H1and Nt DEF19 in tobacco leaves decreased significantly with the increase of nitrogen application rate,and Nt LRR-RK4 also decreased significantly with the extension of leaf infection time.In addition,the expression of these genes in K326 plants infected by A.alternata at the same nitrogen level and at the same time point was significantly higher than that of HD.5,Nitrogen application rate affected the activities of flavonoids and antioxidant enzymes in infected cultivated tobacco.Different doses of nitrogen fertilizer were applied to cultivated tobacco,and then 0 leaves were taken at different time points after inoculation with A.alternata.The results showed that the flavonoids accumulation decreased with the increase of nitrogen application rate,and the flavonoids content in K326 was greater than that in HD at the same time point of the same nitrogen application level.With the increase of nitrogen application,the activity of antioxidant enzyme decreased,and at the same time point of the same nitrogen application level,the activity of antioxidant enzyme in K326 was greater than that in HD.In summary,we found that a potential receptor like kinase gene LRR-RK4 plays an important role in A.alternaria resistance of tobacco.In this study,NaLRR-RK4 regulates the expression of the defense gene NaDEF19 and the synthetase gene NaF6’H1 of plant protection hormone Scopoletin in wild tobacco through the transcription factor NaERF109.In addition,we also found that there were significant differences in the expression of Nt LRR-RK4 among different tobacco varieties,and there were also significant differences in the expression of Nt LRR-RK4 under different nitrogen levels.With the increase of nitrogen application level,the expression of Nt LRR-RK4 in tobacco decreased,and with the decrease of the expression of Nt LRR-RK4,the incidence of tobacco brown spot disease increased.In this paper,we further analyzed the effect of nitrogen fertilizer level on the regulation of antioxidant system by resistance gene Nt LRR-RK4.This paper provides a new idea for the cultivation and selection of varieties resistant to brown spot disease,rational fertilization,and fundamentally solving tobacco brown spot disease.