Cre/loxP, a site-specific recombination system, has been widely used for various purposes, including chromosomal translocations, generation of marker-free transgenic plants, tissue-specific activation of a reporter gene and efficient heterologous gene expression in plants.  However, stable or transient expression of Cre recombinase in plants can cause chlorosis or necrosis.  Here, we describe a modified Cre/loxP recombination system using a DNA fragment flanked with loxP sites in the same orientation in which necrosis induced by Cre recombinase in Nicotiana benthamiana leaves was alleviated.  The modified system was successfully used to create functional GFP-tagged pepper mild mottle virus (PMMoV) and a chimeric virus with coat protein (CP) substitution assembled from separate pro-vector modules.  Our results provide a new strategy and flexible technique to construct chimeric virus and infectious clones for plant viruses with large genomes.

The co-chaperone DnaJ plays an important role in protein folding and regulation of various physiological activities, and participates in several pathological processes.  DnaJ has been extensively studied in many species including humans, drosophila, mushrooms, tomatoes, and Arabidopsis.  However, few studies have examined the role of DnaJ in wheat (Triticum aestivum), and the interaction mechanism between TaDnaJs and plant viruses.  Here, we identified 236 TaDnaJs and performed a comprehensive genome-wide analysis of conserved domains, gene structure and protein motifs, chromosomal positions and duplication relationships, and cis-acting elements.  We grouped these TaDnaJs according to their domains, and randomly selected six genes from the groups for tissue-specific analysis, and expression profiles analysis under hormone stress, and 17 genes for plant virus infection stress.  In qRT-PCR, we found that among the 17 TaDnaJ genes tested, 16 genes were up-regulated after wheat yellow mosaic virus (WYMV) infection, indicating that the TaDnaJ family is involved in plant defense response.  Subsequent yeast two-hybrid assays verified the WYMV NIa, NIb and 7KD proteins interacted with TaDJC (TraesCS7A02G506000), which had the most significant changes in gene expression levels after WYMV infection.  Insights into the molecular mechanisms of TaDnaJ-mediated stress tolerance and sensitivity could inform different strategies designed to improve crop resistance to abiotic and biotic stress.  This study provides a basis for future investigation of the TaDnaJ family and plant defense mechanisms.