We first assessed if the 23 genes up-regulated in elf18 treated EFR:XA21:GFP could be validated by qPCR analysis. Eleven out of 23 DRGs were up-regulated in EFR:XA21:GFP rice leaves after elf18 treatment. Transcripts of the remaining 12 candidate genes were detectable by qPCR amplification but were not up-regulated in elf18 treated EFR:XA21:GFP leaves . We established a detached leaf infection assay to test if genes identified in the EFR:XA21:GFP experiments are representative of genes differentially regulated in Xoo infected Myc:XA21 rice. We observed bacterial ooze from the detached rice leaves three days after inoculation with Xoo strain PXO99A . To further assess if Xoo infects rice leaves in our detached leaf infection assay, we measured the expression level of Os8N3 , which was previously shown to be up-regulated in rice upon Xoo infection and is thus a useful marker of successful infection . For these experiments, we also included a mutant PXO99A strain that is unable to infect rice as a control. The hrpA1 gene encodes a pilus protein that is essential for type IIIsecretion of effectors required for host infection . We observed that the PXO99A1 hrpA1 Xoo mutant is unable to infect Kitaake and Myc:XA21 rice plants . Both WT Kitaake and Myc:XA21 detached leaves express Os8N3 at higher levels compared to mock treatments 24 hpi with WT PXO99A, but not with PXO99A1 hrpA1 .
These results demonstrate that Xoo infects detached rice leaves. We next employed the detached leaf infection assay to examine the expression of the stress-related marker PR10b in Xoo infected Myc:XA21 rice leaves. Compared with mock treated controls,flower bucket PR10b is up-regulated in flg22 treated rice, elf18 treated EFR:XA21:GFP rice and Myc:XA21 rice treated with the RaxX21-sY . Using qPCR, we detected significant up-regulation of PR10b expression in Myc:XA21 rice leaves 24 hpi with PXO99A and PXO99A1 hrpA1. PR10bup-regulation was not observed in infected Kitaake leaves . These results show that the detached leaf infection assay can be used to assess XA21-mediated marker gene expression and also indicate that RaxX expression or secretion is not affected by the 1 hrpA1 mutation. In this study we identified 8 genes that are specifically up-regulated in both elf18 treated EFR:XA21:GFP and Xoo infected detached Myc:XA21 rice leaves. At the time of these experiments, the activator of XA21, RaxX, had not yet been identified . We therefore treated rice plants expressing the EFR:XA21:GFP chimera with elf18 to identify candidate marker genes because EFR:XA21:GFP rice are partially resistant to Xoo and respond to elf18 treatments as described above in the introduction. Our results show that even though the EFR:XA21:GFP-mediated response does not confer robust resistance to Xoo , similar genes are up-regulated during both EFR:XA21:GFP- and Myc:XA21-mediated responses .
Further studies are necessary to determine why the expression of EFR:XA21:GFP in rice does not confer robust resistance to Xoo. We show that stress-related gene induction of PR10b in Myc:XA21 rice leaves is maintained in plants inoculated with PXO99A1 hrpA1 mutant strains. These results suggest that RaxX expression, modification and secretion is not compromised by the 1 hrpA1 mutation. These results indicate that RaxX function is independent of type-III secretion mediated by hrpA1. It was previously reported that the raxSTAB operon, which encoded predicted components of a type-I secretion system, was required for the processing and secretion of the XA21 elicitor . Our finding that RaxX function is independent of hrpA1-mediated type-III secretion is consistent with the hypothesis that RaxX is a type I-secreted molecule and may provide insight into the largely unknown biological function of RaxX. The discovery of RaxX and the establishment of the detached leaf infection assay described here provide useful tools for studying XA21-mediated immunity. XA21 activation can be measured through ROS production and marker gene expression in detached leaves treated with the RaxX21-sY peptide . One advantage of this approach is that researchers can study XA21-mediated immunity without working with Xoo. Instead, researchers can activate XA21-mediated immunity by treating leaves with RaxX21-sY peptide rather than Xoo. This strategy eliminates the need for select agent permits, which are costly and time-consuming.
The assay described in this study now allows researchers to use Xoo infected plants to monitor XA21 activation by gene expression, which was previously only possible using peptide treatments. This provides the benefit of monitoring bacterial induced genes, such as Os8N3 . While we are not able to definitively assess resistance versus susceptibility to Xoo using this assay, we demonstrate that we can use gene expression to monitor an immune response specifically mediated by XA21. The detached leaf infection assay can also be used for other studies of bacterial-rice interactions. For example, this system can be used to study rice immune responses conferred by different resistance genes or induced by different bacterial strains. For example, the detached leaf infection assay can be used to study the immune response conferred by other rice Xa genes that confer resistance to Xoo such as Xa3/Xa26, which also encodes a cell surface receptor kinase . The detached leaf infection assay can also be adapted to study immune responses to other races of Xooor other Xanthomonas pathovars such as Xanthomonas oryzae pv. oryzicola .Throughout every organism’s lifecycle, exposure to a variety of biotic and abiotic stresses can threaten their survival. Unlike motile animals that can relocate to avoid certain threats, plants are sessile organisms that must cope with external stresses to a much greater extent than mobile organisms. During the course of their evolution, the development of complex mechanisms has allowed plants to grow under unfavorable and even hostile conditions. The majority of plant species are rooted in the soil; thus the quality of the soil greatly influences the virility of plants. From the soil, plant roots must absorb sufficient water and mineral nutrients to sustain both their underground and above ground organs. While plants are dependent on the nutrients provided in soils in order to grow, they are adversely affected by undesirable compounds in the soil such as salinity,plastic flower bucket extremes of pH, and toxic substances. Aluminum is a detrimental metal ion in soils around the globe that drastically reduces root growth with a concomitant reduction of shoot growth. This negatively impacts global crop growth due to this failure of the roots to support the above ground organs with the water and nutrients needed in order for the whole plant to thrive. The phytotoxicity of Al is dependent on soil pH, and while Al is abundant in soils all around the globe, Al toxicity is confined to acidic soils. The mechanisms of Al resistance are of great interest to better understand how plants, especially crop species, can grow on Al toxic soils in order to feed a growing global population.Al toxicity is a global agricultural problem and is one of the predominant factors that limit crop productivity in acidic soil regions. Acidic soils have a pH < 5.5 at the surface layer and comprise greater than 30% of the land in the world , with varying estimates having 50-70% of the potentially arable lands in the acidic range . While plant growth inhibition in acidic soils is due to a combination of mineral toxicities as well as nutrient deficiencies, Al toxicity is the single most important factor inhibiting plant growth and the major constraint for crop production . Al is the most abundant metal in the earth’s crust and the third most prevalent element on earth behind oxygen and silicon .
The majority of Al is contained within aluminosilicate compounds in the primary mineral, or as Al oxides and aluminosilicates in secondary minerals, which are formed during weathering . However, Al in these forms is not phytotoxic and only a small percentage of Al in the soil solution contributes to Al toxicity in plants . It is the soluble Al ion, Al3+, that is primarily responsible for plant growth inhibition due to Al toxicity. Al solubility is dependent on the pH of the soil solution; therefore Al toxic soils are confined to acid soil environments. Al can become dissociated from the primary and secondary minerals in acidic conditions, which can add to soil acidity. In acid soils, soluble Al speciates into the toxic trivalent cation, Al3+, at concentrations that rapidly inhibit root growth. As the pH decreases further, the concentration of Al in the soil solution increases. Between pH of 4.0 to 4.5 in the soil solution, a small change in the acidity can cause large changes in the solubility of Al . For example, at pH <4.2, Al3+ can be found at extremely high concentrations ranging from 0.1 to 1.0 mmol/L . Even though Al exists in many forms such as monovalent, divalent and trivalent Al ions, Al oxides, Al sulfates, Al fluorides and the complex Al13, only Al3+ and the Al13 complex have been proven to be toxic to plants . Of these forms, Al3+ is the most prevalent species in soils that are pH < 4.7, and is therefore the most significant form for Al phytotoxicity. As such, from here out the elemental abbreviation Al will represent Al3+ in regards to phytotoxicity unless otherwise specified. Soil acidity is determined by the concentration of hydrogen ions in the soil solution. A variety of factors both natural and manmade affect soil acidity including the innate characteristic of the soil, farming practices, as well as biological and environmental changes. Decay of organic material that forms carbonic acid and other weak acids can add to the acidity of the soil solution . The amount of rainfall can affect the rate of soil acidification depending on the rate of rain water flow through the soil solution . Also, soils that develop from granite materials acidify faster than soils that have developed from calcareous materials. Sandy soils also acidify more rapidly due to higher leaching of alkalinizing agents and reduced buffering capacity . Utilization of high-input farming practices is common in industrialized nations that include the overuse of ammonia-based fertilizers, which cause further acidification of agricultural soils . Over cropping can also lead to the depletion of essential nutrient cations from the soil solution such as calcium, potassium, magnesium and sodium .For example, removal of the straw from wheat cropping depletes basic cations to a great extent and further enhances acidification by nitrification . While the cumulative impact of these factors may be difficult to quantify over the course of a few years, it is important to acknowledge that other possible factors could additionally be contributing to the rapid acidification of the soil solutions. Originally Al toxicity was considered to be restricted to tropical agricultural regions, or areas of high rainfall with highly weathered soils; but soil acidity has now attracted global attention. Acid soils occur primarily in two geographic humidity belts: a cool-climate northern belt spanning North America, Europe, and Northern Asia, and a warm-climate southern tropical belt spanning Central and South America, Sub-Saharan Africa, South East Asia including its islands and Australia . In addition, two thirds of the acid soils are found primarily in forested areas and 17% are covered by prairie, savanna, or steppe vegetation while only 6% of the world’s acid soils are used for agriculture, indicating that acid soils severely compromise agricultural production . Because acid soils are detrimental to crop production, especially several cereal species, the widespread occurrence of acid soils serves as a severe limitation on agricultural production in these geographic belts. Often the practice of liming surface soils is used in industrialized countries to ameliorate soil acidity, and therefore Al toxicity; however this practice is neither affordable nor effective as a long-term practice. The bulk of agricultural lime is derived from limestone but is also sourced from marl, slag from iron and steel making, flue dust from cement plants, and refuse from sugar beet factories, paper mills, calcium carbide plants, rock wool plants, and water softening plants . However total production and use of lime is relatively low and is generally only applied to farming areas near the source, ie industrialized areas. Lime is either sprinkled onto soils to be mixed in during tillage operations or dissolved in water where it hydrolyzes to form –OH ions to raise soil pH, but only at the surface of soils .