YNM158 can be effectively applied in FHB improvement in wheat breeding
Breeding and applying FHB resistant varieties in wheat production is an effective way to control the destructive disease. However, the long-term intraspecific cross breeding of wheat reduced the range of genetic variation among varieties and had poor resistance to FHB, while the related wild species and genera of wheat carry many FHB resistance genes. For example, the FHB resistance genes Fhb3, Fhb6 and Fhb7 on chromosome 7Lr#1S of Leymus racemosus, chromosome 1E(ts)#1S of Elymus tsukushiensis and chromosome 7el2 of Th. ponticum, respectively were reported to have major resistance to FHB [6–9]. In addition, the 1Yc and 3Sc chromosomes of Roegneria ciliaris [21], the 1E, 7E chromosome of diploid Th. elongatum, the chromosome 3St of Elymus repens [22], and the 7Mg chromosome of Aegilops geniculata [23] also possessed the FHB resistance genes. Although the introduction of alien chromosomes can improve the resistance to FHB, it also brings some genetic encumbrance, which makes the agronomic characters of most foreign germplasm poor and difficultly to be directly used in wheat breeding for FHB resistance. Therefore, to make full use of wheat related species in wheat breeding for FHB resistance, it is necessary to create small fragment translocation lines to develop new varieties with increased FHB resistance and no yield penalty. At present, Fhb7 gene from Th. ponticum was poured into cultivated wheat through small segment translocation lines and used in wheat breeding for FHB resistance [9]. The diploid form of tall wheatgrass, Th. elongatum, has a high level of resistance to FHB and was used to increase FHB resistance in wheat cultivar Chinese Spring by translocation development [19, 24]. In this study, we have successfully established the translocation lines with the small fragments of chromosome 7EL from diploid Th. elongatum by physical radiation, one of which has excellent agronomic traits and high resistance to wheat FHB and named YNM158. Different from previous reports, the translocation in YNM158 occurred on chromosome 4BS, a non-compensative translocation, and the reason for this phenomenon was that chromosome translocation was induced by ionizing radiation, the breakage and reconnection of wheat and alien chromosomes were random, so most of them were uncompensated translocations. Interestingly, the survey results of agronomic traits for two consecutive years showed that YNM158 did not have any bad performances due to a non-compensatory translocation line, such as genetic instability of exogenous chromosomes, high plant height and poor fertility. This may be because a small alien chromosome fragment was attached to the distal end of 4BS chromosome in recurrent parent in YNM158, whereas no chromosome fragments were lost in wheat. And previous studies have also shown that non-compensatory translocation lines also have important utility in wheat breeding. For example, the small fragment translocation line 5VS-6AS·6AL can be used to improve the quality of wheat soft grains [25], the 3A-7Js translocation line can be used to improve wheat stem rust resistance [26], two homozygous translocation lines T1AS·1AL-6VS and T4BS·4BL-6VS-4BL carrying Pm21 can be used to enhance powdery mildew resistance in wheat [27]. Therefore, we propose the translocation line YNM158, contained small fragment of 7EL chromosomes, has a good application prospect in the breeding for resistance to FHB in wheat.
Transcriptome analysis validated that glutathione metabolic is one of the important contributors to FHB resistance in pathogen infection stage
Transcriptome analysis is a valuable tool in investigating the molecular mechanisms behind cereal resistance to fungal infections as the costs of this technique decrease and its applications become more widespread. In this study, we found that compared with YM158, the glutathione metabolic was the most significantly enriched pathway in YNM158 after F. graminearum infection during the infection stage (Fig. 5D). It is well known that plants respond to fungal infections by activating defense genes including producing reactive oxygen species (ROS) which can enhance the strength of plant cell wall to resist the invasion and colonization of pathogens [28, 29]. However, when ROS accumulates in large amounts in plants, it will cause oxidative stress, damage plant cells, and lead to cell dysfunction and even death. Currently, the enzymes involved in the antioxidant defense system can be divided into two groups: (i) Enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase GPX, glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR); (ii) Non-enzymatic antioxidants such as ascorbic acid (AA), reduced glutathione (GSH), α-tocopherol, carotenoids, plastoquinone/ubiquinone and flavonoids [30]. Early research reported that ascorbate-glutathione (AsA-GSH) cycle is the important way to diameter of removal of ROS in plant. Among them, APX is the key enzymes of this cycle, which can utilize AsA as the electron donor reducing H2O2 to water, and prevents the accumulation of a toxic level of H2O2 in photosynthetic organisms under stress conditions [31]. And the glutathione (GSH) participate in various metabolic processes and were the essential components of antioxidative and detoxification systems in plant cells [32]. It can be used as both a reducing agent and a strong nucleophile, participating in the elimination of reactive oxygen species (ROS) through thiol-disulphide redox reactions, and in the detoxification of various heterogenic organisms through conjugation reactions, respectively [33]. However, GSH will be oxidized to oxidized glutathione (GSSG) in the reaction of clearing ROS. In order to maintain the balance of GSH content in the plant, GR enzyme will effectively and timely reduce GSSG to GSH. It can be seen that GR enzyme plays a very important role in clearing ROS and maintaining the content of GSH in plants. For example, overexpression of GR gene from Haynaldia villosa in wheat can increase resistance to powdery mildew [34]. In this study, we found that the expression of some APX-encoding and GR-encoding genes in YNM158 was up-regulated during the infection stage (Fig. 5D). Therefore, we suggest that, glutathione may play a key role in ROS-mediated resistance to FHB in wheat.
In addition, glutathione S-transferase (GST) represents a group of multifunctional enzymes widely present in plants and plays important roles in plant secondary metabolism [35], growth and development [36], and biotic and abiotic stress responses [37]. Furthermore, one of its most important functions is the ability to inactivate toxic compounds. Because GST can form complexes with glutathione (GSH) by catalyzing hormones, toxins to inactivate or eliminate toxicity of many substances, and expel them in the body under the action of relevant transporters [38]. These results suggested that GST played a crucial role in plant disease resistance. For example, NbGSTU1 can increase the resistance to Colletotrichum destructivum in Nicotiana benthamiana [39]. The lack of GSTU13 function resulted in enhanced disease susceptibility toward several fungal pathogens in Arabidopsis thaliana [40]. Overexpression of LrGST5 in tobacco can improve the resistance of transgenic plants to F. oxysporum [41]. TaGSTU6 interactions can enhance wheat resistance to powdery mildew but not wheat stripe rust [42]. We know that wheat infected with FHB can be contaminated with a variety of mycotoxins, especially deoxynivalenol (DON) [43]. It has been reported that GSH can form a GSH-DON conjugates under the catalysis of GST to reduce the accumulation of DON and protect plants from toxicity. For instance, Fhb7 and FhbRc1, encoding glutathione S-transferase, enhanced the resistance to FHB in wheat background [9, 44]. In this study, the expression of GST- encoding genes, including TraesCS1A03G0109100, TraesCS3D03G0946300, TraesCS4D03G0493500, TraesCS5B03G0050700, TraesCS5A03G0730500, TraesCS5B03G0770700 and Tel7E01G1020600 was significantly up-regulated after infection with F. graminearum in YNM158. Among them, the expression of the Fhb7 homolog Tel7E01G1020600 increased sharply at 72 hpi, which was tens of times higher than that of the non-infection (Fig. 3E). It can be seen that GST is one of the important contributors to FHB resistance roles in pathogen infection stage. However, the Tel7E01G1020600 in YNM158 was derived from diploid Th. elongatum, which is not consistent with the origin of Fhb7. So, whether Tel7E01G1020600 in YNM158 has the same disease resistance function as Fhb7 needs to be further studied.
Other genes from 7EL fragment in YNM158 might also be involved in increasing FHB resistance especially in pathogen initial colonization stage
DON toxin is a very important fungal pathogen when F. graminearum infects wheat. It can synthesize a large amount of F. graminearum along the inflorescence axis and promote the process of disease expansion. However, some studies have reported that when the pathogen initially infected wheat anthers, there was no DON synthesis signal, and only when the disease spread along the inflorescence axis from the inoculation point, DON began to be synthesized in large quantity in the pathogen [45, 46]. It can be seen that DON can help the pathogen spread along the wheat spike axis, but it is not necessary for its initial infection [47]. In the process of long-term co-evolution between plants and pathogens, a series of complex defense mechanisms have gradually formed. Generally, pathogen-associated molecular pattern (PAMP)-trigged immunity (PTI) is the first defensive line of plant innate immunity and is mediated by pattern recognition receptors (PRRs). And the PRRs are divided into two types, receptor-like kinases (PLKs) and receptor-like proteins (PLPs). To date, many PLKs have been found to play a key role in wheat disease resistance. For example, Sun et al. (2023) reported that a repeat receptor-like kinase-encoding gene TaBIR1 contributed to wheat resistance against Puccinia striiformis f. sp. tritici by mediating ROS production and callose deposition [48], and the cysteine-rich receptor-like kinase TaCRK3 contributed to defense against Rhizoctonia cerealis in wheat through directing antifungal activity and heightening the expression of defense-associated genes in the ethylene signaling pathway [49]. And the RLKs have also been found to contribute to grain resistance to Fusarium resistance in cereals. For instance, Thapa et al. (2018) identified two homologous genes on barley chromosome 6H (HvLRRK-6H) and wheat chromosome 6DL (TaLRRK-6D), respectively, which could enhance cereal resistance to FHB disease [50]. And the Arabidopsis senses Fusarium elicitors in early immune responses to extracts from Fusarium spp. by a novel receptor complex which was encoded by the leucine-rich repeat receptor-like kinase MDIS1-interacting receptor-like kinase 2 (MIK2) at the cell surface [51]. Interesting, we also identified several RLKs-encoding genes on the 7EL fragment in this study, and the expression of them was significantly up-regulated at initial colonization stage after F. graminearum inoculation, such as the expression of Tel7E01G943900 which was significantly up-regulated at 0.5 hpi (Fig. 3F).
In addition, in immune responses, plants have developed a number of disease-resistance mechanisms to resist nutrient uptake by pathogens, which involve sugar transport, metabolism, and signal transduction. The previous studies have shown that hexose released by cell wall invertase (CWIN) activity not only acts as a signal molecule to trigger the expression of disease-resistance related genes, but also is an essential metabolite and energy source for the synthesis of antioxidant compounds and defense molecules, such as salicylic acid and callose [52–54]. For example, AtSTP4 and Atβfruct1 encoding monosaccharide transporter and CWIN, respectively, are both induced in Arabidopsis during parasitic infection by fungus [55]. Chang et al. (2020) reported that silence the hexes transporter-encoding gene PsHXT1 in wheat stripe rust can significantly inhibit the pathogenicity of pathogenic bacteria [56]. In this study, the expression of a monosaccharide-sensing protein-encoding gene Tel7E01G980900 was significantly up-regulated within 8h after infection with F. graminearum and reached the highest level at 2 hpi in YNM158 (Fig. 3G). And knowledge of the function of monosaccharide-sensing protein is similar to that of hexose transporters [57, 58]. Therefore, we speculated that they can also be conducted with CWINs to bring hexose back to host cells, reducing sugar availability to the pathogen, and thus improve host disease resistance. But this needs to be confirmed in the further studies.
It is well known that F. graminearum is a kind of facultative trophic bacteria. Therefore, wheat needs to use a series of defense mechanisms to resist pathogen infection at different stages. The introduction of 7EL chromosome fragments not only brought GST-encoding gene which was one of the important contributors on DON detoxification, but also brought other genes which were up-regulated at initial colonization stage. And these genes were also involved in increasing FHB resistance. Therefore, in-depth study of these genes can provide new insights into the molecular mechanisms of wheat resistance to FHB.
Introgression of 7EL fragment altered the gene expression in wheat after F. graminearum inoculation
The introduction of alien chromosome fragments not only brought the resistance genes, but also affects gene expression on normal chromosomes [59, 60]. In this study, we also found that the translocation of chromosomes affected the expression of wheat genes which were enriched in the resistance pathways including phosphatidylinositol signaling system, protein processing in endoplasmic reticulum, plant-pathogen interaction and MAPK signaling pathway at different stages with F. graminearium infection.
When plants are infected with pathogens, phospholipase C (PLC) is rapidly activated by different pathogen-associated molecular patterns (PAMPs) and effector proteins in plant cells [61]. And then catalyze phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2] to produce inositol 2-phosphate (IP2) or inositol 3-phosphate (IP3) and diacylglycerol (DAG). These are conserved compounds of pathogenic microbes that are perceived by immune receptors present in resistant plants [62, 63]. The previous studies reported that silencing and knock‑out SlPLC2 in tomato can reduce susceptibility to Botrytis cinereal [61, 62]. And the SlPLC6 plays a key role in both for Ve1 resistance protein mediated resistance to Verticillium dahliae and Pto/Prf resistance protein mediated resistance to Pseudomonas syringae [64]. In addition, it has been reported that salicylic acid (SA), jasmonate (JA)and methyl jasmonate can increase the expression of OsPI-PLC in rice (Oryza sativa) and improve the resistance of rice to Magnaporthe oryzae [65]. In this study, it was found that the expression of some PLC genes in YNM158 was higher than that in YN158 at the initial colonization stage, such as TraesCS4A03G0225500 and TraesCS4B03G0547100. Moreover, the results of qRT-PCR showed that the expression of TraesCS4D03G0528700, which encoded phosphatidylinositol 4-phosphate-5 kinase (PIPK5), in YNM158 was higher than that in YM158 at initial colonization stage (Fig. 7B). We know that PIP5K was the catalytic enzyme for the synthesis of PI(4,5)P2 and Shimada et al., (2019) have pointed out that the biosynthesis of PI(4,5)P2 was an important target to improve the defense ability of Arabidopsis thaliana against Colletotrichum, and its activity also determines the defense ability of Arabidopsis thaliana against Colletotrichum [66]. Therefore, we speculated that PIP5K gene can affect the accumulation of PI(4,5)P2 in YNM1158 to participate in the PLC-mediated response to F. graminearium infection, and thus affect the colonization of F. graminearium to improve the resistance to FHB during the initial stages of infection.
There are also defense-related proteins in plants that are synthesized by the rough endoplasmic reticulum (RER), so when plants attacked by the pathogen, the genes encoding endoplasmic reticulum (ER) chaperones are induced, such as the immunoglobulin-binding protein (BIP), heat shock protein (Hsp), calreticulin (CRT) and protein disulfide isomerase (PDI)-encoding genes. The previous studies have shown that the Hsp was one of the ER chaperones, which play an indispensable role as molecular chaperones in the quality control of PRRs and intracellular resistance (R) proteins against potential invaders [67]. For example, Hsp90 was not only involved in the defense of many microbial pathogens by activating the cytosolic R proteins containing nucleotide-binding domain and a leucine-rich repeat, but also participated in chitin responses and anti-fungal immunity in a chaperone complex with its co-chaperone Hop/Sti1 [67, 68]. In terms of specific diseases, cytoplasmic Capsicum annuum Hsp70 (CaHsp70) can enhance the resistance to Xanthomonas campestris pv. vesicatoria in pepper [69], GmHsp40 can increase soybean resistance to Soybean mosaic virus [70], Hsp70 can enhance the resistance to powdery mildew in cucumber under heat shock-induction [71], MeHsp90.9-MeSGT1-MeRAR1 chaperone complex interacted with MeATGs to trigger autophagy signaling to improve disease resistance to cassava bacterial blight [72]. In this study, we found the expression of TraesCS4B03G0573000, which encodes the heat shock protein in YNM158 significantly induced to be upregulated after infection by F. graminearium at initial colonization stage, which was opposite to the expression pattern in YM158 (Fig. 7F). Usually, the expression of the genes encoding ER chaperones even predates the expression of genes encoding pathogenesis-related (PR) proteins [73]. Therefore, we inferred that the expression of genes encoding Hsp protein would rapidly induce after infection with F. graminearium in YNM158, thus activating the defense mechanism earlier, inducing programmed cell death, affecting the colonization of pathogens, and making plants resistant to disease, which provides a new idea for further research on the mechanism of FHB resistance.
Reactive oxygen species (ROS) are the important signaling molecules in defense responses during plant-pathogen interactions, which are mainly produced by respiratory burst oxidase homologs (RBOHs) [74]. In Arabidopsis, AtRBOHD and AtRBOHF are responsible for ROS production against pathogen attacks [75]. In Nicotiana benthamiana, NbRBOHA and NbRBOHB silencing leads to less ROS production and reduced the resistance against the infection by potato pathogen Phytophthora infestans [76]. Phosphorylation is known to be one of the essential mechanisms of RBOHD activation and is also transcriptionally activated by some kinases, such as MAPK cascades, and the transcriptional regulation of RBOHs may play a key roles in subsequent ROS bursts after turnover of the plasma membrane-localized RBOHs used for the first burst [77]. For example, Yamamizo et al. (2007) reported that MAPK kinase was involved in inducing the response of potato StRBOHC and StRBOHD genes in response to pathogen signals in potato [78], and Asai et al. (2008) also illustrated that the MAPK cascade MEK2-SIPK regulates the oxidative burst resulting from the induction of RBOHB expression in resistance to P. infestans and Colletotrichum orbiculare of N. benthamiana [79]. Here, we identified the expression of a RBHO-encoded gene TraesCS1A03G0718100 was up-regulated after F. graminearum infection in YNM158 (Fig. 7J), as well as some genes encoding MAPK kinase (Fig. 5G). Therefore, we hypothesized that MAPK kinase in YNM158 might be involved in inducing RBOH gene response to the resistance against F. graminearum. However, this puzzle requires further investigations.