A new vaccination regimen using adenovirus-vectored vaccine confers effective protection against African swine fever virus in swine.

African swine fever (ASF) is an acute and highly contagious lethal infectious disease in swine that severely threatens the global pig industry. At present, a safe and efficacious vaccine is urgently required to prevent and control the disease. In this study, we evaluated the safety and immunogenicity of replication-incompetent type-2 adenoviruses carrying African swine fever virus (ASFV) antigens, namely CP204L (p30), E183L (p54), EP402R (CD2v), B646L (p72), and B602L (p72 chaperone). A vaccine cocktail delivered by simultaneous intramuscular (IM) and intranasal (IN) administration robustly elicited both systemic and mucosal immune responses against AFSV in mice and swine and provided highly effective protection against the circulating ASFV strain in farmed pigs. This multi-antigen cocktail vaccine was well tolerated in the vaccinated animals. No significant interference among antigens was observed. The combined IM and IN vaccination using this adenovirus-vectored antigen cocktail vaccine warrants further evaluation for providing safe and effective protection against ASFV infection and transmission.

Ethylene enhances MdMAPK3-mediated phosphorylation of MdNAC72 to promote apple fruit softening.

The phytohormone ethylene plays an important role in promoting the softening of climacteric fruits, such as apples (Malus domestica); however, important aspects of the underlying regulatory mechanisms are not well understood. In this study, we identified apple MITOGEN-ACTIVATED PROTEIN KINASE 3 (MdMAPK3) as an important positive regulator of ethylene-induced apple fruit softening during storage. Specifically, we show that MdMAPK3 interacts with and phosphorylates the transcription factor NAM-ATAF1/2-CUC2 72 (MdNAC72), which functions as a transcriptional repressor of the cell wall degradation-related gene POLYGALACTURONASE1 (MdPG1). The increase in MdMAPK3 kinase activity was induced by ethylene, which promoted the phosphorylation of MdNAC72 by MdMAPK3. Additionally, MdPUB24 functions as an E3 ubiquitin ligase to ubiquitinate MdNAC72, resulting in its degradation via the 26S proteasome pathway, which was enhanced by ethylene-induced phosphorylation of MdNAC72 by MdMAPK3. The degradation of MdNAC72 increased the expression of MdPG1, which in turn promoted apple fruit softening. Notably, using variants of MdNAC72 that were mutated at specific phosphorylation sites, we observed that the phosphorylation state of MdNAC72 affected apple fruit softening during storage. This study thus reveals that the ethylene-MdMAPK3-MdNAC72-MdPUB24 module is involved in ethylene-induced apple fruit softening, providing insights into climacteric fruit softening.

Exogenous Ca2+ promotes transcription factor phosphorylation to suppress ethylene biosynthesis in apple.

Ethylene biosynthesis in apple (Malus domestica) fruit can be suppressed by calcium ions (Ca2+) during storage; however, the underlying mechanisms are unclear. In this study, we identified the apple transcription factor MCM1-AGAMOUS-DEFICIENS-SRF5 (MdMADS5), which functions as a transcriptional activator of the ethylene biosynthesis-related gene 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE1 (MdACS1), a partner of the calcium sensor CALCIUM-DEPENDENT PROTEIN KINASES7 (MdCDPK7). Ca2+ promoted the MdCDPK7-mediated phosphorylation of MdMADS5, which resulted in the degradation of MdMADS5 via the 26S proteasome pathway. MdCDPK7 also phosphorylated 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE1 (MdACO1), the key enzyme in ethylene biosynthesis, leading to MdACO1 degradation and inhibition of ethylene biosynthesis. Our results reveal that Ca2+/MdCDPK7-MdMADS5 and Ca2+/MdCDPK7-MdACO1 are involved in Ca2+-suppressed ethylene biosynthesis, which delays apple fruit ripening. These findings provide insights into fruit ripening, which may lead to the development of strategies for extending the shelf life of fruit.

Phosphorylation of MdCYTOKININ RESPONSE FACTOR4 suppresses ethylene biosynthesis during apple fruit ripening.

The plant hormone ethylene plays a central role in the ripening of climacteric fruits, such as apple (Malus domestica). Ethylene biosynthesis in apple fruit can be suppressed by calcium ions (Ca2+); however, the underlying mechanism is largely unknown. In this study, we identified an apple APETALA2/ETHYLENE-RESPONSIVE FACTOR (AP2/ERF) transcription factor, MdCYTOKININ RESPONSE FACTOR4 (MdCRF4), which functions as a transcriptional activator of ethylene biosynthesis- and signaling-related genes, including Md1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE1 (MdACS1) and MdETHYLENE-RESPONSIVE FACTOR3 (MdERF3), as a partner of the calcium sensor, calmodulin. Ca2+ promoted the Ca2+/CaM2-mediated phosphorylation of MdCRF4, resulting in MdCRF4 recognition by the E3 ubiquitin ligase MdXB3 ORTHOLOG 1 IN ARABIDOPSIS THALIANA (MdXBAT31), and consequently its ubiquitination and degradation via the 26S proteasome pathway. This in turn resulted in lower expression of MdACS1 and MdERF3 and reduced ethylene biosynthesis. Transiently overexpressing various MdCRF4 proteins with specific mutated phosphorylation sites revealed that the phosphorylation state of MdCRF4 affects the ripening of apple fruit. The results reveal that a Ca2+/CaM-MdCRF4-MdXBAT31 module is involved in Ca2+-suppressed ethylene biosynthesis, which delays apple fruit ripening. This provides insights into fruit ripening that may result in strategies for extending fruit shelf life.

NUCLEOCYTOPLASMIC shuttling of ETHYLENE RESPONSE FACTOR 5 mediated by nitric oxide suppresses ethylene biosynthesis in apple fruit.

Nitric oxide (NO) is known to modulate the action of several phytohormones. This includes the gaseous hormone ethylene, but the molecular mechanisms underlying the effect of NO on ethylene biosynthesis are unclear. Here, we observed a decrease in endogenous NO abundance during apple (Malus domestica) fruit development and exogenous treatment of apple fruit with a NO donor suppressed ethylene production, suggesting that NO is a ripening suppressor. Expression of the transcription factor MdERF5 was activated by NO donor treatment. NO induced the nucleocytoplasmic shuttling of MdERF5 by modulating its interaction with the protein phosphatase, MdPP2C57. MdPP2C57-induced dephosphorylation of MdERF5 at Ser260 is sufficient to promote nuclear export of MdERF5. As a consequence of this export, MdERF5 proteins in the cytoplasm interacted with and suppressed the activity of MdACO1, an enzyme that converts 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. The NO-activated MdERF5 was observed to increase in abundance in the nucleus and bind to the promoter of the ACC synthase gene MdACS1 and directly suppress its transcription. Together, these results suggest that NO-activated nucleocytoplasmic MdERF5 suppresses the action of ethylene biosynthetic genes, thereby suppressing ethylene biosynthesis and limiting fruit ripening.

Auxin-activated MdARF5 induces the expression of ethylene biosynthetic genes to initiate apple fruit ripening.

The gaseous plant hormone ethylene induces the ripening of climacteric fruit, including apple (Malus domestica). Another phytohormone, auxin, is known to promote ethylene production in many horticultural crops, but the regulatory mechanism remains unclear. Here, we found that auxin application induces ethylene production in apple fruit before the stage of commercial harvest, when they are not otherwise capable of ripening naturally. The expression of MdARF5, a member of the auxin response factor transcription factor (TF) family involved in the auxin signaling pathway, was enhanced by treatment with the synthetic auxin naphthaleneacetic acid (NAA). Further studies revealed that MdARF5 binds to the promoter of MdERF2, encoding a TF in the ethylene signaling pathway, as well as the promoters of two 1-aminocyclopropane-1-carboxylic acid synthase (ACS) genes (MdACS3a and MdACS1) and an ACC oxidase (ACO) gene, MdACO1, all of which encode key steps in ethylene biosynthesis, thereby inducing their expression. We also observed that auxin-induced ethylene production was dependent on the methylation of the MdACS3a promoter. Our findings reveal that auxin induces ethylene biosynthesis in apple fruit through activation of MdARF5 expression.