Huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus worldwide. HLB is associated with the non-culturable bacterium, Candidatus Liberibacter asiaticus (CaLas) in the United States. The virulence mechanism of CaLas is largely unknown, partly because of the lack of a mutant library. In this study, Tobacco mosaic virus (TMV) and Nicotiana benthamiana (N. benthamiana) were used for large-scale screening of the virulence factors of CaLas. Agroinfiltration of 60 putative virulence factors in N. benthamiana led to the identification of four candidates that caused severe symptoms in N. benthamiana, such as growth inhibition and cell death. CLIBASIA_05150 and CLIBASIA_04065C (C-terminal of CLIBASIA_04065) could cause cell death in the infiltrated leaves at five days post infiltration. Two low-molecular-weight candidates, CLIBASIA_00470 and CLIBASIA_04025, could inhibit plant growth. By converting start codon to stop codon or frameshifting, the four genes lost their harmful effects to N. benthamiana. It indicated that the four virulence factors functioned at the protein level rather than at the RNA level. The subcellular localization of the four candidates was determined by confocal laser scanning microscope. CLIBASIA_05150 located in the Golgi apparatus; CLIBASIA_04065 located in the mitochondrion; CLIBASIA_00470 and CLIBASIA_04025 distributed in cells as free GFP. The host proteins interacting with the four virulence factors were identified by yeast two-hybrid. The host proteins interacting with CLIBASIA_00470 and CLIBASIA_04025 were overlapping. Based on the phenotypes, the subcellular localization and the host proteins identified by yeast two-hybrid, CLIBASIA_00470 and CLIBASIA_04025, functioned redundantly. The hypothesis of CaLas virulence was proposed. CaLas affects citrus development and suppresses citrus disease resistance, comprehensively, in a complicated manner. Ubiquitin-mediated protein degradation might play a vital role in CaLas virulence. Deep characterization of the interactions between the identified virulence factors and their prey will shed light on HLB. Eventually, it will help in developing HLB-resistant citrus and save the endangered citrus industry worldwide.

Growth regulatory factors (GRF) are plant-specific transcription factors that play pivotal roles in growth and various abiotic stresses regulation. However, adaptive evolution of GRF gene family in land plants are still being elucidated. Here, we performed the evolutionary and expression analysis of GRF gene family from seven representative species. Extensive phylogenetic analyses and gene structure analysis revealed that the number of genes, QLQ domain and WRC domain identified in higher plants was significantly greater than those identified in lower plants. Besides, dispersed duplication and WGD/segmental duplication effectively promoted expansion of the GRF gene family. The expression patterns of GRF gene family and target genes were found in multiple floral organs and abundant in actively growing tissues. They were also found to be particularly expressed in response to various abiotic stresses, with stress-related elements in promoters, implying potential roles in floral development and abiotic stress. Our analysis in GRF gene family interaction network indicated the similar results that GRFs resist to abiotic stresses with the cooperation of other transcription factors like GIFs. This study provides insights into evolution in the GRF gene family, together with expression patterns valuable for future functional researches of plant abiotic stress biology.

Background: Potato (Solanum tuberosum) is the fourth most important food crop in the world and plays an important role in food security. Drought stress has a significantly negative impact on potato growth and production. There are several publications involved drought stress in potato, this research contributes to enrich the knowledge. Results: In this study, next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing technology were used to study the transcription profiles in potato in response to 20%PEG6000 simulates drought stress. The leaves of the variety “Désirée” from in vitro plantlets after drought stress at six time points from 0 to 48 hours were used to perform NGS and SMRT sequencing. According to the sequencing data, a total of 12,798 differentially expressed genes (DEGs) were identified in six time points. The real-time (RT)-PCR results are significantly correlated with the sequencing data, confirming the accuracy of the sequencing data. Gene ontology and KEGG analysis show that these DEGs participate in response to drought stress through galactose metabolism, fatty acid metabolism, plant-pathogen interaction, glutathione metabolism and other pathways. Through the analysis of alternative splicing of 66,888 transcripts, the functional pathways of these transcripts were enriched, and 51,098 transcripts were newly discovered from alternative splicing events and 47,994 transcripts were functionally annotated. Moreover, 3445 lncRNAs were predicted and enrichment analysis of corresponding target genes was also performed. Additionally, Alternative polyadenylation was analyzed by TADIS, and 26,153 poly (A) sites from 13,010 genes were detected in the Iso-Seq data. Conclusion: Our research greatly enhanced potato drought-induced gene annotations and provides transcriptome-wide insights into the molecular basis of potato drought resistance.