The biological activity of plant polysaccharides can be enhanced by sulfated modification. In this study, the immunomodulatory effect of sulfated Cyclocarya paliurus polysaccharides (SCP3) on macrophages RAW264.7 and its potential molecular mechanism were investigated. Results showed that SCP3 at 25-100 μg/mL increased viability and improved phagocytosis of RAW264.7 cells. Meanwhile, SCP3 could activate MAPK and NF-κB signaling pathways, which increased the phosphorylation of Erk1/2, JNK, p38 and NF-κB p65, promoting secretion of cytokines TNF-α, IL-6 and NO as well as the production of ROS. In addition, TLR4 receptor inhibitors were able to block the production of NO and TNF-α by SCP3-stimulated macrophages. Based on western blot analysis and validation using specific inhibitors against MAPK and NF-κB signaling pathways, the results demonstrated that SCP3 induced macrophages activation and enhanced TNF-α and NO production via TLR4-mediated MAPK and NF-κB pathways. In summary, SCP3 has significant immunomodulatory potential. The underlying molecular mechanism was that SCP3 activates macrophages via TLR4 receptors to promote ROS production, which in turn activates the downstream MAPK/NF-κB signaling pathway and then increases the secretion levels of cytokines and NO.

Copper (Cu)-based antimicrobial compounds (CBACs) have been widely used to control phytopathogens for nearly fourteen decades. Since the first commercialized Bordeaux mixture was introduced, CBACs have been gradually developed from highly to slightly soluble reagents and from inorganic to synthetic organic, with nanomaterials being a recent development. Traditionally, slightly soluble CBACs form a physical film on the surface of plant tissues, separating the micro-organisms from the host, then release divalent or monovalent copper ions (Cu²⁺ or Cu⁺) to construct a secondary layer of protection which inhibits the growth of pathogens. Recent progress has demonstrated that the release of a low concentration of Cu²⁺ may elicit immune responses in plants. This supports a triple-tiered protection role of CBACs: break contact, inhibit microorganisms, and stimulate host immunity. This spatial defense system, which is integrated both inside and outside the plant cell, provides long-lasting and broad-spectrum protection, even against emergent copper-resistant strains. Here, we review recent findings and highlight the perspectives underlying mitigation strategies for the sustainable utilization of CBACs.

Xanthomonas oryzae delivers transcription activator-like effectors (TALEs) into plant cells to facilitate infection. Following economic principles, the redundant TALEs are rarely identified in Xanthomonas. Previously, we identified the Tal2b, which activates the expression of the rice 2-oxoglutarate-dependent dioxygenase gene OsF3H_03g to promote infection in the highly virulent strain of X. oryzae pv. oryzicola HGA4. Here, we reveal that another clustered TALE, Tal2c, also functioned as a virulence factor to target rice OsF3H_04g, a homologue of OsF3H_03g. Transferring Tal2c into RS105 induced expression of OsF3H_04g to coincide with increased susceptibility in rice. Overexpressing OsF3H_04g caused higher susceptibility and less salicylic acid (SA) production compared to wild-type plants. Moreover, CRISPR–Cas9 system-mediated editing of the effector-binding element in the promoters of OsF3H_03g or OsF3H_04g was found to specifically enhance resistance to Tal2b- or Tal2c-transferring strains, but had no effect on resistance to either RS105 or HGA4. Furthermore, transcriptome analysis revealed that several reported SA-related and defense-related genes commonly altered expression in OsF3H_04g overexpression line compared with those identified in OsF3H_03g overexpression line. Overall, our results reveal a functional redundancy mechanism of pathogenic virulence in Xoc in which tandem Tal2b and Tal2c specifically target homologues of host genes to interfere with rice immunity by reducing SA.

Downy Mildew Resistance 6-like (DMR6-like) genes are identified as salicylic acid (SA) hydroxylases and negative regulators of plant immunity. Previously, we identified two rice DMR6-like genes, OsF3H03g, and OsF3H04g, that act as susceptible targets of transcription activator-like effectors (TALEs) from Xanthomonas oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak (BLS) in rice. Furthermore, all four homologs of rice DMR6-like proteins were identified to predominantly carry the enzyme activity of SA 5-hydroxylase (S5H), negatively regulate rice broad-spectrum resistance, and cause the loss of function of these OsDMR6s, leading to increased resistance to rice blast and bacterial blight (BB). Here, we curiously found that an OsF3H04g knock-out mutant created by T-DNA insertion, osf3h04g, was remarkedly susceptible to BLS and BB and showed an extreme reduction in SA content. OsF3H04g knock-out rice lines produced by gene-editing were mildly susceptible to BLS and reduced content of SA. To explore the susceptibility mechanism in OsF3H04g loss-of-function rice lines, transcriptome sequencing revealed that another homolog, OsS3H, had induced expression in the loss-of-function OsF3H04g rice lines. Furthermore, we confirmed that a great induction of OsS3H downstream and genomically adjacent to OsF3H04g in osf3h04g was primarily related to the inserted T-DNA carrying quadruple enhancer elements of 35S, while a slight induction was caused by an unknown mechanism in gene-editing lines. Then, we found that the overexpression of OsS3H increased rice susceptibility to BLS, while gene-editing mediated the loss-of-function OsS3H enhanced rice resistance to BLS. However, the knock-out of both OsF3H04g and OsS3H by gene-editing only neutralized rice resistance to BLS. Thus, we concluded that the knock-out of OsF3H04g activated the expression of the OsS3H, partially participating in the susceptibility to BLS in rice.