Endo-type alginate lyases usually degrade alginate completely into various size-defined unsaturated oligosaccharide products (≥disaccharides), while exoenzymes primarily produce monosaccharide products including saturated mannuronate (M) and guluronate (G) units and particularly unsaturated Δ units. Recently, two bifunctional alginate lyases have been identified as endolytic but M- and G-producing with variable action modes. However, endolytic Δ-producing alginate lyases remain undiscovered. Herein, a new Flammeovirga protein, Aly2, was classified into the polysaccharide lyase 7 superfamily. The recombinant enzyme and its truncated protein showed similar stable biochemical characteristics. Using different sugar chains as testing substrates, we demonstrated that the two enzymes are bifunctional while G-preferring, endolytic whereas monosaccharide-producing. Furthermore, the catalytic module of Aly2 can vary the action modes depending on the terminus type, molecular size, and M/G content of the substrate, thereby yielding different levels of M, G, and Δ units. Notably, the enzymes preferentially produce Δ units when digesting small size-defined oligosaccharide substrates, particularly the smallest substrate (unsaturated tetrasaccharide fractions). Deletion of the non-catalytic region of Aly2 caused weak changes in the action modes and biochemical characteristics. This study provided extended insights into alginate lyase groups with variable action modes for accurate enzyme use.

Arabidopsis BOTRYTIS-INDUCED KINASE1 (BIK1) is areceptor-like cytoplasmic kinase acting early in multiple signaling pathways important for plant growth and innate immunity. It is known to form a signaling complex with a cell-surface receptor FLS2 and a co-receptor kinase BAK1 to transduce signals upon perception of pathogen-associated molecular patterns (PAMPs). Although site-specifi c phosphorylation is speculated to mediate the activation and function of BIK1, few studies have been devoted to complete profiling of BIK1 phosphorylation residues. Here, we identified nineteen in vitro autophosphorylation sites of BIK1 including three phosphotyrosine sites, thereby proving BIK1 is a dual-specifi city kinase for the fi rst time. The kinase activity of BIK1 substitution mutants were explicitly assessed using quantitative mass spectrometry (MS). Thr-237, Thr-242 and Tyr-250 were found to most signifi cantly affect BIK1 activity in autophosphorylation and phosphorylation of BAK1 in vitro. A structural model of BIK1 was built to further illustrate the molecular functions of specifi c phosphorylation residues. We also mapped new sites of FLS2 phosphorylation by BIK1, which are different from those by BAK1. These in vitro results could provide new hypotheses for more in-depth in vivo studies leading to deeper understanding of how phosphorylation contributes to BIK1 activation and mediates downstream signaling specifi city.

Polygalacturonase-inhibiting proteins (PGIPs) have been shown to recognize fungal polygalacturonases (PGs), which initiate innate immunity in various plant species. Notably, the connection between rice OsPGIPs and PGs in Xanthomonas oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak (BLS), remains unclear. Here, we show that OsPGIP1 was strongly induced after inoculating rice with the Xoc strain RS105. Furthermore, OsPGIP1-overexpressing (OV) and RNA interference (RNAi) rice lines increased and decreased, respectively, the resistance of rice to RS105, indicating that OsPGIP1 contributes to BLS resistance. Subsequently, we generated the unique PG mutant RS105Δpg, the virulence of which is attenuated compared to that of RS105. Surprisingly, the lesion lengths caused by RS105Δpg were similar to those caused by RS105 in the OV lines compared with wild-type ZH11 with reduced Xoc susceptibility. However, the lesion lengths caused by RS105Δpg were still significantly shorter in the OV lines than in ZH11, implying that OsPGIP1-mediated BLS resistance could respond to other virulence factors in addition to PGs. To explore the OsPGIP1-mediated resistance, RNA-seq analysis were performed and showed that many plant cell wall-associated genes and several MYB transcription factor genes were specifically expressed or more highly induced in the OV lines compared to ZH11 postinoculation with RS105. Consistent with the expression of the differentially expressed genes, the OV plants accumulated a higher content of jasmonic acid (JA) than ZH11 postinoculation with RS105, suggesting that the OsPGIP1-mediated resistance to BLS is mainly dependent on the plant cell wall-associated immunity and the JA signaling pathway.

Background: Plant viruses cause severe economic losses in agricultural production. An ultrahigh activity plant immune inducer (i.e., ZhiNengCong, ZNC) was extracted from endophytic fungi, and it could promote plant growth and enhance resistance to bacteria. However, the antiviral function has not been studied. Our study aims to evaluate the antiviral molecular mechanisms of ZNC in tobacco. Results: Here, we used Potato X virus (PVX), wild-type tobacco and NahG transgenic tobacco as materials to study the resistance of ZNC to virus. ZNC exhibited a high activity in enhancing resistance to viruses and showed optimal use concentration at 100-150 ng/mL. ZNC also induced reactive oxygen species accumulation, increased salicylic acid (SA) content by upregulating the expression of phenylalanine ammonia lyase (PAL) gene and activated SA signaling pathway. We generated transcriptome profiles from ZNC-treated seedlings using RNA sequencing. The first GO term in biological process was positive regulation of post-transcriptional gene silencing, and the subsequent results showed that ZNC promoted RNA silencing. ZNC-sprayed wild-type leaves showed decreased infection areas, whereas ZNC failed to induce a protective effect against PVX in NahG leaves. Conclusion: All results indicate that ZNC is an ultrahigh-activity immune inducer, and it could enhance tobacco resistance to PVX at low concentration by positively regulating the RNA silencing via SA pathway. The antiviral mechanism of ZNC was first revealed in this study, and this study provides a new antiviral bioagent.