Alfalfa is a perennial leguminous forage grass that occupies an important position in animal husbandry because of its high yield and quality. It is widely used in feed production. In China, with the implementation of a series of policies to support the cultivation of alfalfa and other forage products, the alfalfa industry has achieved unprecedented development, with the planting area continuing to expand. However, many alfalfa diseases seriously restrict the healthy development of the alfalfa industry. The most representative is alfalfa root rot (Fusarium root rot) (Fang et al., 2019; Guo et al., 2022). According to statistics, the annual loss of alfalfa production caused by root rot worldwide is approximately 20%, and in severe cases, it is as high as 40%. Alfalfa root rot generally occurs in the main alfalfa planting areas in northwest China (Gansu, Xinjiang, Qinghai, etc.), northern China (Hebei and Inner Mongolia, etc.), and northeast China (Heilongjiang and Jilin, etc.), and the mortality rate of alfalfa in severe fields is > 60% (Li-xia et al., 2008; Pan et al., 2015). Alfalfa root rot is widely distributed and destructive and is one of the main factors leading to a decline in alfalfa yield and quality deterioration (Gao, 2024). This disease is usually caused by a combination of soil fungi, among which Fusarium fungi are the main pathogens causing root rot in alfalfa fields (Li et al., 2019). The incidence of Fusarium root rot in alfalfa has recently increased. To date, 25 species of Fusarium have been reported to cause root rot in alfalfa. There are eight main Fusarium species causing alfalfa root rot in China (Fang et al., 2019), namely, Fusarium oxysporum, Fusarium solani, Fusarium acuminatum, Fusarium equiseti, Fusarium semitectum, Fusarium avenaceum, Fusarium proliferatum, and Fusarium tricinctum. In recent years, it has been reported in China that Fusarium incarnatum, Fusarium redolens, Fusarium verticillioides, Fusarium falciforme, and Fusarium virguliforme can also cause alfalfa root rot (Wang et al., 2023; Yang et al., 2024). Therefore, it is of great significance to study the molecular mechanisms of alfalfa in response to Fusarium infection to formulate effective prevention and control strategies for alfalfa root rot.

During Fusarium infestations, plants produce complex physiological responses. For example, previous studies have found that proteins associated with photosynthesis are mostly inhibited, whereas pathways involved in energy metabolism, protein synthesis, and conversion are often upregulated (Cong et al., 2017; Wang et al., 2024). In these studies, the plants also exhibited other characteristic changes, including to their glucose metabolism, cell wall, reactive oxygen species (ROS), increased enzyme activity, expression of pathogenicity-related (PR) proteins, and secondary metabolites (Kang et al., 2019; Fang et al., 2023). Wang’s results indicate that after inoculation with Fusarium, the levels of soluble glucose, soluble protein, and malondialdehyde (MDA) in alfalfa plants changed significantly. The cytochrome P450, MYB, ERF, NAC, and bZIP protein families are also considered to be closely related to the resistance of alfalfa to root rot (Wang et al., 2023). In an experiment in which kidney bean roots were inoculated with F. oxysporum, transcriptome and metabolome analyses showed that the salicylic acid, jasmonic acid, and ethylene pathways were significantly enriched, with the enrichment of the flavonoid biosynthesis pathway being the most significant (Chen et al., 2019). In transgenic alfalfa plants with down-regulated expression of Caffeoyl-CoA O-methyltransferase(CCoAOMT) gene, the resistance to Fusarium wilt was enhanced. Transcriptomic and metabolomic analyses showed that the down-regulation of CCoAOMT led to the accumulation of (iso)flavonoids and intermediate compounds in their pathway, thereby improving the disease resistance of alfalfa (Gill et al., 2018). Protein expression plays an important role in plant responses to environmental stress and infections. A quantitative proteomic analysis of two rice genotypes 93-11 and Nipponbare, with different levels of disease resistance after inoculation with Fusarium fujikuroi, suggested that aquaporin PIP2-2 may play an antagonistic role against rice blast disease (Ji et al., 2019). The roots and leaves of alfalfa inoculated with F. proliferatum were analyzed by proteomics, revealing 66 DEPs in the roots and 27 DEPs in the leaves. These identified proteins were classified into different categories involving defense and stress response-related metabolism, photosynthesis, and protein synthesis (Cong et al., 2017).

Inner Mongolia, one of the main alfalfa planting areas in China, occupies an important position in the development of the alfalfa industry. The author collected alfalfa root rot samples from four major alfalfa-producing areas in Inner Mongolia, China, and identified the dominant pathogen as F. acuminatum (Sun, 2023). When Wang isolated and identified alfalfa root rot in different regions of Inner Mongolia, he found that the dominant pathogen was F. acuminatum (Wang, 2023). In recent years, several studies have shown that F. acuminatum can cause root rot in a variety of plants, including angelica, lentils, peppercorns, and astragalus (Zhang, 2021; Ruan et al., 2022; Bugingo et al., 2024; Mu et al., 2024). Current studies on F. acuminatum mostly focus on the isolation and identification of pathogens, determination of their pathogenicity and biological characteristics, evaluation of resistance to various pathogens, and biological control. The mechanism of the interaction between F. acuminatum and host plants has rarely been reported. Using alfalfa inoculated with F. acuminatum as experimental materials, we analyzed the changes in gene and protein expressions of alfalfa during the infection process of F. acuminatum using transcriptome and proteome sequencing. In the present study, we aimed to explore the molecular mechanisms of alfalfa resistance to F. acuminatum and identify candidate genes associated with resistance or susceptibility, which will not only provide a new perspective for the study of the molecular mechanisms underlying alfalfa resistance to Fusarium root rot but also establish a valuable genetic basis for the breeding of alfalfa disease-resistant varieties and contribute to the healthy and sustainable development of China’s alfalfa industry.

Plants have evolved complex stress-resistance mechanisms to cope with harsh natural conditions as they grow (Glazebrook, 2005; Höfte and Voxeur, 2017). The plant cell wall is the first physical barrier against pathogen invasion and is often thought to be a passive barrier initiated during pathogen invasion. However, an increasing number of studies have suggested that the cell wall may actively play a defensive role (Scheller and Ulvskov, 2010; Wan et al., 2021). Cell walls undergo structural changes as plants grow, develop, and respond to stress. As an important monitoring system, they undergo dynamic remodeling during adaptation to external stress from pathogens. Thus, maintaining cell wall integrity (CWI) is important in activating and monitoring plant defense responses (Zhao and Dixon, 2014; Vaahtera et al., 2019). In pathogen-associated molecular pattern-triggered immunity (PTI), the host exhibits cell wall thickening, lignification, phytoprotective hormone production, and PR gene expression (Liu et al., 2018). In addition, cellulose, dextrin, galacto-oligosaccharide, xylo-oligosaccharides, and substances released when the cell wall is infected with pathogens can act as effective elicitors or signaling molecules to enhance plant disease resistance (Wan et al., 2021). Zhao conducted a comprehensive analysis using metabolomics, proteomics, and transcriptomics to identify the genes affecting the total lignin content in Populus tomentosa. Through GO and KEGG analyses of lignin-related modules, it was found that the total lignin content is influenced not only by individual lignin genes but also by genes involved in pathways such as “glutathione metabolic process,” “cellular modified amino acid metabolic process,” and “carbohydrate catabolic process” (Zhao et al., 2023). Glutathione peroxidase (GSH-PX) is involved in ROS and lignin metabolism and can significantly affect the content of total phenols as a precursor of lignin synthesis, as well as phenylalanine aminolyase (monolignol biosynthesis), thus affecting the total lignin content (Juan-juan et al., 2011). In the present study, the GO analysis of alfalfa DEGs showed that most of the alfalfa DEGs were enriched in the cell cortex, cell wall organization, and cell wall during infection with F. acuminatum. The results of the KEGG enrichment analysis showed that the DEGs were mainly enriched in glutathione metabolism (mtr00480), including 25 genes that were upregulated and 6 genes that were downregulated. Glutathione metabolism was related to the lignin content, indicating that these genes may play a role in cell wall morphological changes by affecting lignin synthesis. Pectin is an important component of the cell wall (Wan et al., 2021), and methylesterases can directly or indirectly participate in defense reactions by regulating the degree of the methyl esterification of pectin. Previous proteomics studies on F. oxysporum-infected resistant and susceptible chickpea genotypes have shown that methylesterase levels increased in both chickpea varieties 72 h after inoculation. However, methylesterase accumulated to a greater extent in the resistant varieties (Chatterjee et al., 2014). Similarly, in the proteomic analysis of this study, it was found that the methyl esterase (G7K9E3) in alfalfa was significantly upregulated 3 days after pathogen inoculation. The GO analysis of DEGs in the transcriptome also showed that numerous genes were enriched in O-methyltransferase activity. The accumulation of methyl esterase may potentially be involved in cell wall repair. These results show that lignin and pectin, which are important components of the cell wall, play important defensive roles when alfalfa is infection by F. acuminatum.

Glucose in plants not only serves as an essential component for energy supply and structural building blocks but also possesses signal transduction functions, enabling it to regulate the expression of related genes and the activity of enzymes (Proels and Hückelhoven, 2014; He et al., 2016). Glucose can directly act as an antioxidant to quench oxidative substances (Kunz et al., 2014) and participate in plant growth and development, as well as adverse environmental responses (Lastdrager et al., 2014; Lu et al., 2015). Numerous studies have revealed the crucial role of glucose in the plant response to pathogenic stress. Wang et al. (2023) found that after inoculation with F. acuminatum, the soluble glucose content in both disease-resistant and disease-susceptible alfalfa varieties increased significantly, with the content in the resistant variety being approximately twice that in the susceptible one (Wang et al., 2023). Similarly, in the response of chickpeas to F. oxysporum, proteins involved in glycolysis showed an upward trend in their expression in disease-resistant varieties and a downward trend in susceptible varieties, indicating that the activation and rapid accumulation of sugars affect the sensitivity of chickpeas to pathogenic bacteria (Kumar et al., 2016). In addition, an omics analysis of F. oxysporum infection in common beans revealed that the amino sugar and nucleotide sugar metabolic pathways were significantly enriched (Chen et al., 2019). Collectively, these studies indicate that glucose metabolism is closely associated with plant disease resistance. The present study further elucidated the role of glucose in alfalfa resistance against F. acuminatum using omics analyses. The results showed that the proteins involved in the glycolytic pathway were significantly enriched. Trehalose, a typical stress metabolite, was significantly upregulated in the glycolytic pathway. Trehalose usually does not accumulate under suitable conditions but rapidly accumulates under stress (Tang et al., 2018; Wang et al., 2022). In plants, trehalose is synthesized via the TPS-TPP pathway (Vandesteene et al., 2012). In-depth analyses revealed that trehalose-6-phosphate phosphatase (TPP) was significantly upregulated among the DEPs involved in the glycolysis/glucose metabolism pathway. The GO analysis showed that the trehalose-6-phosphate (T6P) protein (A0A072U2F7) in the trehalose biosynthetic process was also significantly upregulated. Notably, previous studies have indicated that the trehalose metabolic pathway, which is regulated solely by the expression of trehalose-6-phosphate synthase (TPS), can only lead to a small amount of trehalose accumulation, which is insufficient to enhance plant resistance to osmotic stress. It has been speculated that a slight increase in trehalose may be a response to adverse stress as a signaling substance (Bischof, 2020). Other studies have shown that in the trehalose metabolic pathway, the change in the T6P content is more crucial for enhancing plant stress resistance (Paul et al., 2018). Previous studies on trehalose have mostly focused on aspects such as the abiotic stress response of plants and regulation of flowering time (Du et al., 2023). Combined with the fact that no significant upregulation of the TPS gene was detected in the transcriptome in the present study, but both the TPP and T6P proteins were significantly upregulated in the proteome, it is speculated that in the process of alfalfa coping with F. acuminatum stress, it is not trehalose itself but T6P that plays a dominant role in the trehalose metabolic pathway.

When plants are stressed by pathogenic bacteria, the antioxidant enzyme system indirectly enhances disease resistance by eliminating ROS. In this study, the antioxidant and defense-related enzymes (SOD, CAT, APX, and LOX) in alfalfa at different stages of inoculation were examined, including their activities, expression levels of their encoding genes, and protein abundance. The results showed that the activities of the SOD, CAT, APX, and LOX enzymes increased significantly after 3 days inoculation and decreased after 13 days inoculation. The expression of the gene encoding SOD (MS. gene013412) increased significantly after 3 days inoculation and decreased after 13 days inoculation. In contrast, the expression of CAT (MS. gene064838) and APX (MS. gene057684 and MS. gene071408) continued to increase after 3 days inoculation. Although the expression of the related genes changed, there was no significant difference in their protein expression. It has been speculated that this phenomenon may be caused by a post-transcriptional regulatory mechanism. As the main ROS scavenger in plants under stress, As a major ROS scavenger in plants under stress, SOD was abundantly expressed at 3 days after alfalfa inoculation with F. acuminatum. It can catalyze the disproportionation of reactive oxygen species and free radicals to generate H₂O₂ (Vandesteene et al., 2012). With the continuous accumulation of H₂O₂ during infection, APX and CAT are required to further convert it into H₂O₂ and O₂, thereby reducing the toxicity of H₂O₂ to alfalfa cells and maintaining intracellular redox balance (Bischof, 2020). LOX, a class of versatile dioxygenases, plays a unique role in plant defense systems. It can catalyze the oxygenation of polyunsaturated fatty acids to produce hydroperoxides. Through the hydroperoxidation of linoleic and linolenic acids, precursors for jasmonic acid synthesis are produced (Moraga-Suazo et al., 2024), which, in turn, trigger the expression of plant defense-related genes (Yang et al., 2019). This study found that 12 LOX genes were significantly expressed in alfalfa, of which 9 were upregulated and 3 were downregulated. Among the corresponding proteins, three (G7KYM9 was upregulated, G7LIY0 was downregulated, and G7LIZ7 was downregulated) showed significant changes in expression, while the others showed no significant differences. Notably, the three significantly expressed LOX enzymes are involved in linoleic acid metabolism (map00591). Previous studies have shown that LOX expression in wheat is induced by rust disease and may be involved in the effector-triggered immunity (ETI) response (Gan et al., 2024). Maize ZmLOX3 inhibits the colonization and sporulation of Aspergillus flavus. While the maize LOX3 gene defends against the invasion of Aspergillus flavus, the LOX genes in Aspergillus flavus also participate in the host infection process and interact with the host LOX to participate in the host’s resistance and susceptibility to pathogenic fungi. LOX genes are important signal communication molecules in the interaction between the host and pathogenic fungi (Liu, 2016). In a previous study, the leaves of Arabidopsis thaliana and Medicago sativa overexpressing MtLOX24 were infected with Pseudomonas syringae pathovar tomato DC3000 and Fusarium chlamydosporum. The degree of damage to the overexpression group was significantly less than that to the wild-type group (Xu, 2024). Therefore, the role of LOX in alfalfa resistance to F. acuminatum requires further in-depth study.

We further observed the significant enrichment of various receptor protein kinases and heat shock proteins (HSPs). Transcriptome analyses identified 61 HSP genes (27 upregulated and 34 downregulated) and 55 calcium-dependent protein kinase (CDPK) genes (7 upregulated and 48 downregulated). Thirteen HSPs (two HSP90s, two HSP70s, six HSP20s, and two other HSPs) and 20 CDPKs were identified through a proteomic analysis, of which three CDPKs were significantly expressed. HSPs are an important class of highly conserved and abundant molecular chaperones in most organisms. They exert a natural protective effect against various stressors by regulating the establishment of the tertiary structure of proteins, assembly of protein complexes, and transmembrane transport of proteins after processing (Jiang et al., 2023). During plant disease resistance, the interactions among HSP90, SGT1, and RAR1 play a crucial role in stabilizing the activity of R proteins and enabling plants to recognize pathogens. SGT1 interacts with the ATPase region and C terminus of HSP90 via its CS and TPR regions, respectively. The complex formed by these three components plays a central role in the plant disease resistance response triggered by R proteins (Jacob et al., 2017), including RPM1 (resistance to Pseudomonas fluorescens) (Bao et al., 2014), RPS2, and RPM (resistance to Pseudomonas putida in Arabidopsis thaliana) (Takahashi et al., 2003). In this omics analysis, although many HSPs were expressed, their corresponding protein expression levels were relatively low. A similar phenomenon was observed in Arabidopsis thaliana inoculated with Pseudomonas syringae, wherein the HSP90 gene was highly expressed, but the change in protein levels was not significant. According to a previous study, this may have been because plants synthesize SGT1 and HSP90 to cope with stress (Takahashi et al., 2003). In this study, the expression of SGT1 and RAR1 was detected simultaneously; however, the expression of RAR1 was not significantly different. Previous studies have shown that the expression of RAR1 is low under stress and does not respond strongly to pathogenic infection, whereas SGT1 is highly expressed upon infection or stress (Azevedo et al., 2006; Shirasu, 2009). This difference provides insights for a deeper understanding of plant disease resistance mechanisms mediated by HSPs.

CDPKs are serine/threonine protein kinases widely present in plants and protozoa, and they are important calcium (Ca²⁺) sensor proteins. In the plant immune defense system, Ca²⁺ signals are essential for both the PTI and ETI responses. CDPKs possess specific binding motifs, EF-hands, and an N-terminal catalytic kinase domain, which enables them to directly bind to Ca²⁺ or exert their functions by phosphorylating substrate proteins (Gao et al., 2014). Numerous studies have shown that the interactions between CDPKs and their target proteins or substrates can regulate various immune responses. These include the production of ROS during PTI and ETI, regulation of the expression of immune-related genes, and the hypersensitive response triggered by ETI (Freymark et al., 2007; Geng et al., 2013). For example, the wheat calcium-dependent protein kinase 2 (TaCDPK2) is a key protein involved in wheat resistance to powdery mildew. Moreover, the overexpression of TaCDPK2 in rice can significantly enhance the resistance to bacterial blight (Geng et al., 2013). Through transcriptomic and proteomic analyses, we observed the abundant transcripts and protein expression of CDPKs in alfalfa. Among them, three CDPK proteins (A0A072UWP5, A0A072V1D7, and G7J881) were significantly expressed after 13 days inoculation. These proteins were found to be involved in the plant-pathogen interaction (map04626) pathway. Based on the mechanisms of action of CDPKs, it is speculated that these proteins may be activated by Ca²⁺ signals when alfalfa is under pathogenic stress. By regulating downstream substrates, they participate in a complex defense response network and play a crucial role in the resistance of alfalfa to pathogens.

To elucidate the response mechanism of alfalfa to F. acuminatum. This study conducted a combined analysis of alfalfa inoculated with Ustilago maydis at 0, 3, and 13 days using transcriptomics and proteomics. After 3 days inoculation, DEGs and DEPs were mainly enriched in pathways related to the cell cortex, flavonoid biosynthesis, and amino acid metabolism. By day 13, the genes were primarily associated with the cell wall, defense response, and flavonoid biosynthesis.

The study also revealed that metabolic pathways, including secondary metabolites, flavonoids, glycolysis/gluconeogenesis, and plant-pathogen interactions, were significantly enriched at both the mRNA and protein levels, suggesting that these pathways play a key role in the alfalfa response to pathogen stress. During the defense process, the cell wall serves as the first barrier, and its key components, lignin and pectin, play critical roles. Genes involved in lignin biosynthesis and cell wall repair contribute to pathogen defense.

Additionally, antioxidant and defense-related enzymes (SOD, CAT, APX, and LOX) exhibited increased activity during the early stages of infection but decreased activity at later stages. These enzymes play essential roles in scavenging ROS and generating precursors for jasmonic acid synthesis, with LOX being particularly important in alfalfa resistance. HSPs and CDPKs were also significantly enriched. HSPs help protect plants from stress, whereas CDPKs regulate immune responses, with some potentially activated by Ca²⁺ signaling to modulate downstream defense mechanisms.

This study provides new insights into the molecular mechanisms underlying the response of alfalfa to F. acuminatum and offers a valuable basis for identifying candidate genes for breeding alfalfa that are resistant to root rot.