Peptide treatments were performed in the morning and collected at the indicated times

The larger genome size of S. habrochaites suggests the possibility that the putative loss of function of genes and/or genetic elements in S. lycopersicum may be due to deletions or non-functional null mutations. Matsuba et al. sequenced a functional gene cluster for terpene biosynthesis on chromosome 8 of S. habrochaites acc. 1778 and identified several rearrangements, deletions, and a novel gene when compared to the same gene cluster on chromosome 8 of the S. lycopersicum reference genome. Our prior research suggests that the inability of cultivated tomato to maintain shoot turgor under root chilling is the result of a loss of function in S. lycopersicum . Taken together, the current evidence suggests that the S. habrochaites allele for high-resolution mapped QTL stm9 may not be completely syntenic to S. lycopersicum, and that it may not contain the same genic compliment as the S. lycopersicum allele for stm9. Therefore, although the S. lycopersicum genome sequence is helpful in identifying potential candidate genes for shoot turgor maintenance under root chilling, the genomic sequence of the stm9 region of S. habrochaites is necessary for accurate, well-informed candidate gene identification.Stability of QTL expression for tolerance to abiotic stresses is important for successful deployment of stress tolerance QTL in breeding crop plants. Although a significant Genotype × Season interaction was identified for QTL stm9,procona flower transport containers the potential causes of the interaction suggest that this region would likely be useful as a stable source of root chilling tolerance for breeding. A number of other QTL have been identified as targets for breeding despite a significant Genotype × Season interaction in several species, including barley, rice, and maize .

The phenotypic plasticity likely contributed by the stm9 flanking regions suggest that any future breeding strategies should be undertaken with the smallest introgression possible that still contains the entire high-resolution mapped QTL stm9. The S. habrochaites introgression in sub-NIL C7 contains only the high resolution QTL stm9 region . This sub-NIL was grouped as tolerant in both the Spring and Fall datasets, and gave a consistently low stmscore in both seasons , suggesting it may serve as a suitable potential donor parent source of tolerance to root chilling in breeding programs. Due to the complexity of the abiotic stress response pathway, it is unlikely that the S. habrochaites QTL stm9 allele contains only a single gene conferring shoot turgor maintenance under root chilling. Single causal genes have been identified for a number of major QTL , but other major QTL have been shown to be controlled by two or more causal genes or polymorphisms . Identification and testing of the causal gene or polymorphisms underlying QTL stm9 for tolerance to root chilling will be an important step in the identification of genetic targets for improving stress tolerance of plants exposed to root chilling and other types of water stress through marker assisted breeding. Determination of the gene/polymorphisms responsible for a quantitative trait phenotype is facilitated by genomic approaches . Once a target region is identified via high-resolution mapping, a combination of genomic sequencing, structural genomic analysis, and transcriptome profiling can be used to assist in the identification of candidate genes. Therefore a biologically informed ranking of candidate genes located within the QTL stm9 region will require a combination of S. habrochaites genome sequence for this region as well as transcription profiles for susceptible and tolerant subNILs exposed to root chilling.

It is hoped that a better understanding of the underlying mechanism for tolerance to rapid-onset water stress in wild tomato S. habrochaites may aid in the identification of chilling tolerance genes in other species of tropical and sub-tropical origin.Plant immunity is mediated, in part, by cell surface immune receptors that recognize molecules produced by microbes. For example, the Arabidopsis FLS2 and EFR receptors recognize the flg22 peptide derived from bacterial flagellin and the elf18 peptide derived from elongation factor thermounstable protein, respectively . The rice XA21 receptor recognizes the sulfated RaxX peptide derived from the RaxX protein produced by Xanthomonas oryzae pv. oryzae. XA21, EFR, and FLS2 all contain extracellular leucine rich repeat , transmembrane, and intracellular non-RD kinase domains. These receptor domains are partially interchangeable. For example, the LRR domain from EFR can be fused to the transmembrane and intracellular domain of FLS2 to form a chimeric receptor that responds to elf18 treatments when transiently expressed in Nicotiana benthamiana and Arabidopsis thaliana . The EFR LRR can be fused to the transmembrane and intracellular domain of XA21 to form achimeric receptor that responds to elf18 treatment and confers partial resistance to Xoo in transgenic rice lines . The availability of rapid and reliable assays that measure markers characteristic of immune response activation can help facilitate investigations of innate immune signaling. For example, immune signaling studies of FLS2 and EFR in Arabidopsis have been aided by the availability of rapid and reliable assays . In contrast, studies of the XA21-mediated immune response have been limited by the lack of rapid assays and well-characterized genetic markers. Typically, disease assessments are carried out by measuring lesions on rice leaves or by assessing bacterial populations from infected leaves . In this study we aimed to establish a rapid and efficient assay to monitor the XA21- mediated immune response after bacterial infection. For this purpose, we employed the EFR:XA21:GFP chimera composed of the EFR extracellular domain and the XA21 transmembrane and intracellular kinase domains, tagged with green fluorescent protein .

EFR:XA21:GFP transgenic rice plants are partially resistant to Xoo and detached EFR:XA21:GFP leaves respond to elf18 with stress related gene induction, mitogen-activated protein kinase cascade activation, and reactive oxygen species production . These results indicate that plants expressing the EFR:XA21:GFP chimeric protein are appropriate for studies to identify markers of resistance. We used RNA sequencing to identify genes differentially regulated in elf18 treated EFR:XA21:GFP rice. We then assessed if differentially regulated genes in elf18 treated EFR:XA21:GFP rice leaves were up-regulated in Xoo infected rice leaves expressing full-length XA21, which are resistant to Xoo. We developed a rapid and reliable assay to analyze gene expression in detached rice leaves inoculated with Xoo. We identified 8 DRGs from elf18 treated EFR:XA21:GFP rice that are also specifically up-regulated in detached XA21 rice leaves infected with Xoo.For peptide treatments, wild type Kitaake and progeny from line EFR:XA21:GFP-3-8 Kitaake rice leaves were harvested from plants grown in the greenhouse for 4.5 weeks . 1.5–2 cm leaf sections were collected from expanded adult leaves using surgical grade scissors. Thissue from the leaf base and leaf tip was discarded. Detached leaves were equilibrated overnight in 6-well Costar cell culture plates under constitutive light . For bacterial inoculations, we used detached rice leaves harvested from 4-week old plants grown using a hydroponic growth system as described previously under a light intensity of 280 µmol/. Freshly harvested leaves from Kitaake and Ubi-Myc:XA21 Kitaake rice  were cut into 1.5–2 cm pieces and immediately floated on 10 mM MgCl2 solution for mock treatments or 10 mM MgCl2 containing fresh Xoo cell suspensions at O.D.600 of 0.1 . The samples were left overnight under constitutive light and collected 24 h post-inoculation . Leaves were floated on approximately 1.5 mL Xoo cell suspension media in 6-well Corning Costar cell culture plates . The detached leaf infection assay allows a more uniform distribution,procona valencia compared to the scissor inoculation method , of Xoo inoculum by floating leaves on bacterial suspensions. Detached leaves were frozen in liquid nitrogen and powdered using a Qiagen tissue lyser. For tissue from greenhouse grown plants, RNA was extracted from powdered tissue using TRI Reagent and precipitated with isopropanol. For tissue from hydroponically grown plants, RNA was extracted using the Spectrum Plant Total RNA Kit from Sigma-Aldrich. RNA was DNase treated using the TURBO DNase kit from Life Technologies. RNA concentrations were normalized to the lowest sample concentration in each experiment. cDNA was synthesized from 2 µg of total RNA using the High Capacity cDNA Reverse Transcription Kit by Life Technologies. Gene expression changes were determined by 11 Ct method normalized to Actinand compared to mock treated samples. Plant growth, leaf tissue isolation, and treatments were performed as described above. RNA was isolated from untreated Kitaake as well as untreated and elf18 treated EFR:XA21:GFP leaf tissue using the Spectrum Plant Total RNA Kit from Sigma-Aldrich and on-column DNase treated to remove genomic DNA contamination following the manufacturer’s instructions. RNA was quantified using the Quant-IT Ribogreen RNA Assay Kit. Sequences were deposited to the NCBI Sequence Read Archive . RNAseq libraries, sequencing, and reference alignment were performed as described previously . Sample correlation between Kitaake and EFR:XA21:GFP replicates and differential gene expression analysis was performed using the Bioconductor ‘edgeR’ package for R .We generated a PXO99A1 hrpA1 mutant in Philippine race 6 strain PXO99Az, a derivative of strain PXO99 . Xoo was grown in 10 g PSB , 10 g Sucrose, 1 g sodium glutamate , final volume 1L, pH 7.0 or on PSA plates at 28 ◦C.

PXO99A1 hrpA1 was generated by single crossover mutagenesis using the suicide vector pJP5603 . An approximately 500 base pair sequences within hrpA1 was amplified using forward primer 50 -CGGGGTACCGTGCTGCGTGATTTGTCCG-30 and reverse primer 50 – CGCGGATCCTGACTTGGTCGATGCAGTCC-30 and cloned into the multiple cloning site of pJP5603 using the restriction enzyme sites KpnI and BamHI. PXO99A-competent cells were transformed with the suicide plasmids by electroporation and plated to PSA with kanamycin . PXO99A1 hrpA1 colonies with kanamycin resistance were screened by PCR for colonies with single crossover events, which contain the vector disrupting the target gene. PXO99A1 raxST and PXO99A1 raxST complemented strains used in this study were described previously . PXO99A1 raxST evades XA21- mediated immunity while the complemented PXO99A1 raxST strain does not.We analyzed the transcriptomic profile of EFR:XA21:GFP rice lines treated with elf18 to identify genes differentially regulated during this response. We sequenced cDNA from EFR:XA21:GFP leaves treated with 500 nM elf18 for 0.5, 1, 3, 6, and 12 h. We also included untreated EFR:XA21:GFP and Kitaake as controls . Multidimensional scaling of pairwise biological coefficient of variance comparisons for each sample revealed that replicate samples group together . This grouping of biological replicates demonstrates the overall transcriptional similarity between each sample . We identified 2,212 genes that were differentially regulated in EFR:XA21:GFP rice treated with elf18 compared with untreated samples. Using a false discovery rate cutoff of 0.05 and absolute expression log fold change of 2 or greater, we previously reported that the transcriptomic profile of untreated Kitaake compared to untreated EFR:XA21:GFP did not differ significantly . Over the treatment time course, we identified 2,212 DRGs using untreated EFR:XA21:GFP at 0 h as a reference. The number of DRGs that overlap between the elf18 treatment time points are summarized in Fig. 1B and File S1. Of the 2,212 differentially regulated genes, there were 1,420 up-regulated and 792 down-regulated genes. The highest number of DRGs was observed 6 h post elf18 treatment. These results show that elf18 treated EFR:XA21:GFP rice express a substantially different set of genes over time compared to untreated samples. To examine the types of biological processes affected in elf18 treated EFR:XA21:GFP rice, we analyzed GO term enrichment of DRGs using the AgriGo analysis tool . A total of 1,204 out of 1,420 of the up-regulated DRGs and 682 of the 806 down-regulated DRGs had GO annotations. An FDR of 0.05 or less was used to define significantly enriched terms compared to the Michigan State University annotation reference as calculated by the AgriGo tool . Fig. 1C and File S2 summarize the most enriched GO terms in each of the three major DRG clades. Clade 1 contains 1,333 genes that are mostly up-regulated over time. Genes from clade 1 are enriched for metabolic process , response to stimulus and response to stress GO terms . Clade 2 genes are up-regulated across all time points and are enriched for secondary metabolic process , metabolic process and response to stress GO terms . Clade 3 consists of 757 genes that are mostly down-regulated in all time points. Photosynthesis and response to abiotic stimulus are the most enriched GO terms associated with clade 3 genes . We chose 23 DRGs from the elf18 treated EFR:XA21:GFP rice RNAseq dataset with relatively high logFC and low FDR values after 3, 6, and 12 h for detailed analysis.