Isolation And Characterization Of Two Candidate Bacterial Brown Spot Disease Resistance Genes In Maize And Transcriptome Analysis Of A Maize Near-isogenic Line

Bacterial brown spot (BBS) in maize is a seed-borne disease, caused by Pseudomonas syringae pv. syringae Van Holl (Pss). In the present study, we chose the genes Psyl and Psy2that encoding two receptor-like protein kinases (RLKs) as the top candidate genes. Here, we report the cloning and identification of the two candidate resistance genes using the resistant inbred line F349and susceptible inbred line P25. Meanwhile, we have performed the comparative analysis and exploring of the transcriptome in seedling stage of resistant and susceptible near-isogenic lines (NILs) under the seed-borne Pss infection to reveal the resistance mechanism of BBS. The main results are as follows:1. According to the results of fine-mapping, we took the adjacent BACs (AC188128and AC191128) as the reference for genes prediction. Ten EST-supporting predicted genes was found, among which the proteins of Psyl and Psy2have the conserved resistance domain of plant protein kinases.2. The cloning results showed that a13.5Kb huck retrotransposon was inserted within the intron of Psyl gene, a10Kb unknown complex transposon was inserted in the promoter of Psyl gene. However, the insertion of exogenous fragments did not make Psyl gene inactivation. The recessive gene psyl encodes a non-founctional protein which lost the serine/threonine protein kinases active-site. Two recessive genes psy2-1and psy2-2was discovered in susceptible inbred line P25, among which psy2-1gene encodes a non-functional protein for premature transcription termination. The analysis of RNA-Seq data indicate that only psy2-2gene can be expressed. The psy2-2gene has two amino acid mutations within the transmembrane domain, we infer that this is the reason of functional distinctions of encoding proteins between the dominant and recessive genes.3. Bioinformatics analysis indicate that the encoding proteins of Psyl and Psy2possess four protein domains, including B-lectin、S_locus_glycop、PAN_AP_plant and RLK domain. The genes Psyl and Psy2are belong to secreted proteins, and have transmembrane domain leading to the location of plasma membrane.4. Both Psyl and Psy2are constitutively expressed. In the seedling stage of near-isogenic plants, the analysis of expression pattern showed that when the expression levels of two dominant candidate genes were higher than recessive gene before the onset of disease; as the development of plant disease, the expression of recessive genes gradually increased in susceptible NIL. The results of the RNA interference (RANi) showed that Psy2a-RNAi transgenic plants in the seedling stage appeared some severe lesions even develop into adjacent to each other, with similar symptoms to P25, except that the lesions distributed throughout the foliage; The onset of symptoms are not significantly different in Psyi-RNAi and Psy2α-RNAi transgenic plants compared with the control HI II at the adult-plant stage, it is worth noting that Psy2α-RNAi transgenic plants are a few lesions similar to those caused by Pss infection.5. RNA-Seq analysis indicated that Pss infection leads to a sophisticated transcriptional reprogramming in both resistant and susceptible NILs, but defense responses appear temporally different. A number of essential components functional in plant basal resistance were substantially activated, including PEN1, PEN2, PEN3and EDR1, implying that the basal resistance system might be principally active, however, in a basic manner, such resistance is not sufficient to fulfill the complete function to suppress Pss pathogenesis in the susceptible NIL. The expression of a large number of PTI-, ETI-, PR-and WRKY-related genes was pronouncedly activated, suggesting that maize employ a multitude of defense pathways to defend Pss infection. Six R-gene homologs were identified to have significantly higher expression levels in the resistant NIL, indicating that a robust surveillance system (gene-to-gene model) might operate in maize during Pss attack. In some compatible interactions, susceptible plants may exhibit various defense responses similar to R gene-mediated resistance, while these reactions respond temporally slow and unable to inhibit pathogen infection effectively.6. Because of using a common three-dimensional modeling template BAK1, we suppose that maize bacterial browen spot candidate resistance genes Psyl and Psy2similar to tomato bacterial leaf spot resistance gene Pto have the common mechanisms of resistance.