Changes in microbial community structure are widely known to occur after soil amendment with low-molecular-weight organic compounds; however, there is little information on concurrent changes in soil microbial functional diversity and enzyme activities, especially following sorbitol and mannitol amendment. Soil microbial functional diversity and enzyme activities can be impacted by sorbitol and mannitol, which in turn can alter soil fertility and quality. The objective of this study was to investigate the effects of sorbitol and mannitol addition on microbial functional diversity and enzyme activities. The results demonstrated that sorbitol and mannitol addition altered the soil microbial community structure and improved enzyme activities. Specifically, the addition of sorbitol enhanced the community-level physiological profile (CLPP) compared with the control, whereas the CLPP was significantly inhibited by the addition of mannitol. The results of a varimax rotated component matrix demonstrated that carbohydrates, polymers, and carboxylic acids affected the soil microbial functional structure. Additionally, we found that enzyme activities were affected by both the concentration and type of inputs. In the presence of high concentrations of sorbitol, the urease, catalase, alkaline phosphatase, β-glucosidase, and N-acetyl-β-d-glucosaminidase activities were significantly increased, while invertase activity was decreased. Similarly, this increase in invertase, catalase, and alkaline phosphatase and N-acetyl-β-d-glucosaminidase activities was especially evident after mannitol addition, and urease activity was only slightly affected. In contrast, β-glucosidase activity was suppressed at the highest concentration. These results indicate that microbial community diversity and enzyme activities are significantly affected by soil amendment with sorbitol and mannitol.

The recognition of pathogen effectors through the nucleotide-binding leucine-rich repeat receptor (NLR) family is an important component of plant immunity. In addition to typical domains such as TIR, CC, NBS, and LRR, NLR proteins also contain some atypical integrated domains (IDs), the roles of which are rarely investigated. Here, we carefully screened the soybean (Glycine max) genome and identified the IDs that appeared in the soybean TNL-like proteins. Our results show that multiple IDs (36) are widely present in soybean TNL-like proteins. A total of 27 Gm-TNL-ID genes (soybean TNL-like gene encoding ID) were cloned and their antiviral activity towards the soybean mosaic virus (SMV)/tobacco mosaic virus (TMV) was verified. Two resistance (R) genes, SRA2 (SMV resistance gene contains AAA_22 domain) and SRZ4 (SMV resistance gene contains zf-RVT domain), were identified to possess broad-spectrum resistance characteristics towards six viruses including SMV, TMV, plum pox virus (PPV), cabbage leaf curl virus (CaLCuV), barley stripe mosaic virus (BSMV), and tobacco rattle virus (TRV). The effects of Gm-TNL-ID^X (the domain of the Gm-TNL-ID gene after the TN domain) on the antiviral activity of a R protein SRC7^TN (we previously reported the TN domain of the soybean broad-spectrum resistance gene SRC7) were validated, and most of Gm-TNL-ID^X inhibits antiviral activity mediated by SRC7^TN, possibly through intramolecular interactions. Yeast-two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that seven Gm-TNL-ID^X interacted with SMV-component proteins. Truncation analysis on a broad-spectrum antiviral protein SRZ4 indicated that SRZ4^TIR is sufficient to mediate antiviral activity against SMV. Soybean cDNA library screening on SRZ4 identified 48 interacting proteins. In summary, our results indicate that the integration of IDs in soybean is widespread and frequent. The NLR-ID toolkit we provide is expected to be valuable for elucidating the functions of atypical NLR proteins in the plant immune system and lay the foundation for the development of engineering NLR for plant-disease control in the future.

Plant viral diseases cause severe yield losses in global crop production. SRC7, a novel soybean TIR-NBS-BSP-type resistance (R) protein, confers broad-spectrum antiviral immunity against multiple viruses including soybean mosaic virus (SMV), tobacco mosaic virus (TMV), potato virus Y (PVY), and plum pox virus (PPV). Its TIR-NBS domain (SRC7^TN) mediates full antiviral resistance, making it an ideal bait for screening immune-related proximal candidate proteins. In this study, TurboID-based proximity labeling (PL) was applied to profile the SRC7^TN proximal candidate proteins in N. benthamiana. We identified 262 high-confidence proximal candidate proteins, which were mainly enriched in plant immune signaling, redox regulation, protein modification and nuclear-related pathways. Among them, 21 candidates were functionally characterized, revealing multiple host factors contributing to SRC7-dependent antiviral immunity. Eleven interactions were confirmed via bimolecular fluorescence complementation (BiFC), and two nucleoside/nucleotide hydrolase-family proteins MZN14.5 and RIHA were subjected to further interaction-domain analysis. Collectively, this work constructs a high-confidence SRC7^TN proximal-interaction network and highlights critical host factors in SRC7-mediated broad-spectrum antiviral immunity. It also validates TurboID’s utility for screening for NLR-proximal candidate proteins in heterologous plant systems and provides insights into R-protein-governed plant antiviral defense.

Defense responses against pathogens are essential for plant growth, and the nucleotide-binding leucine-rich repeat receptor (NLR) family is an important component of plant immunity. Besides NBS and LRR domains, NLR proteins also contain TIR, CC, or RPW8 domain at their N-terminus and the functions of these canonical domains are well documented. Recently, some atypical X domains, also named integrated domains (IDs), have been reported to be encoded by NLR genes and to play key roles in effector-triggered immunity. Here, we screened the plant NLR database to identify atypical NLRs in potato. A total of 181 atypical NLR protein entries, identified from 642 candidates as truncated or St-NLR-IDs (potato NLRs encoding ID) and comprising 147 NLR-IDs and 34 truncated NLRs, were classified into four major groups, including T(TIR)-class, C(CC)-class, N(NBS)-class, and X-class, and together with 12 typical CNL/TNL sequences, they were used for phylogenetic analysis. Promoter regions of the majority of these entries contain “stress and defense” elements such as MYB/MYC cis-regulatory element, indicating their involvement in plant immune response. In the Rywal background, the expression of six genes was upregulated and that of nineteen genes was downregulated. In the NahG-Rywal plants, the expression of seventeen genes was upregulated and that of eight genes was downregulated. In the shRBOHD-Rywal background, the expression of eighteen genes was upregulated and that of six genes was downregulated. We also investigated the expression of four C-JID-containing NLR-ID genes, M1BEE2, M1BF53, M1A5Z0, and M1BZB1, upon PVX, PVY infection and SA treatment by RT-qPCR and suggested their potential involvement in plant immunity. Promoter–GUS assays for the above four genes also supported their responses to viral infection and SA treatment. In summary, we identified and classified atypical NLRs in potato, analyzed their expression under PVY^N-Wilga infection using public transcriptome data, and validated the virus- and SA-responsive expression of four C-JID-encoding genes, providing a resource for future functional studies.