## 3 Discussion  
In this study, we initially analyzed the transcriptome of _P. eucalypt_ FBS135 in response to nitrogen scarcity. The RNA-Seq result showed that a total of 1936 genes changed significantly upon nitrogen starvation. The expression levels of genes related to nitrogen source metabolism and nitrogen source utilization were mostly downregulated, and the expression levels of genes related to ABC transporters, microbial metabolism in diverse environments, two-component systems, and ribosome pathways in different environments were most significantly changed. Expression changes of all the genes at the transcription level and their expression patterns represented a possible general fitness mechanism of these kinds of endophytes to nitrogen limitation. Furthermore, we identified four genes that may be related to the low-nitrogen adaption process through constructing and screening a transposon library, including the gene for the host-specific protein J, RNA polymerase \(\sigma\) factor RpoS, phosphoribosamine-glycine ligase, and serine acetyltransferase. These genes provide molecular targets for further investigations of the tolerance of the endophytes to nitrogen starvation. Endophytes are natural components of plant microecosystems. Due to their close relationship with plants, they have more direct and effective impact on plants than other associated microorganisms. However, the mechanism of how endophytic bacteria improve the resistance of host plants against various stresses is still poorly understood. Many endophytes, including some _Pantoea_ species, have the ability to fix nitrogen [35; 36; 37]. The bacteria can convert molecular nitrogen into ammonia ions through the action of nitrogenase. The fixed nitrogen can be finally absorbed and utilized by the host plants, as shown in sugarcane, corn, rice, wheat, and other crops [35; 36; 37; 45; 46; 47; 48]. In recent years, some researchers also demonstrated that nitrogen-fixing endophytes in woody plants such as pine trees also act as and provide an additional nitrogen source, helping plants to form enormous amounts of biomass in barren soil [49; 50; 51; 52]. In previous work, we isolated the endophyte _P. eucalypt_ FBS135 from pine trees and demonstrated its growth-promoting effect on pine [41]. The ability of FBS135 to grow on/in a chemically defined medium with a lack of nitrogen source led us to speculate that FBS135 was able to fix nitrogen, which, however, was negated experimentally later [42]. However, a new question has thus been raised concerning how endophytes like FBS135 adapt to an extremely low supply of nitrogen in some environmental niches. Exploring the underlying mechanism may have great significance for understanding the relationship between endophytes and their host plants. Here we identified that a large number of _P. eucalypt_ FBS135 genes altered their transcription under the condition of NH\({}_{4}\)+ nitrogen. Their expression patterns were linked to a stringent response to nitrogen starvation, which at least includes a general decreased expression of genes, typically those such as the ribosome-related genes. This change may reduce the nitrogen demand for some protein production and other unnecessary biological processes. Only a small number of genes were upregulated including those involved in transportation, which may promote the bacterial ability to acquire nitrogen from environments, and some genes for organonitrogen compound biosynthesis and metabolism. The regulation of the genes seems logical for the response of a bacterium to an environment lacking enough nitrogen supply. However, more principles and details could be scrutinized to understand the adaption mechanism of FBS135 to the stress of nitrogen limitation. The changed expression of many FBS135 genes may be associated with the gene _rpoS_, which we identified using the transposon library. According to the transcriptome data, _rpoS_ was upregulated by approximately threefold upon nitrogen deficiency.