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Angustifolius lupine (NLL, Lupinus angustifolius L.) is a leguminous plant used for food production and soil improvement. The global expansion of NLL as a crop has attracted many pathogenic fungi, including lupine anthracnose, which causes the devastating anthracnose disease. Two alleles, Lanr1 and AnMan, which confer increased resistance, have been used in NLL breeding, but the underlying molecular mechanisms remain unknown. In this study, the Lanr1 and AnMan markers were used to screen European NLL samples. Testing of the vaccine in a controlled environment confirmed the efficacy of both resistant donors. Differential gene expression profiling was performed on representative resistant and susceptible lines. Anthracnose resistance was associated with overexpression of the gene ontology terms “GO:0006952 Defense Response”, “GO:0055114 Redox Process”, and “GO:0015979 Photosynthesis”. In addition, the Lanr1(83A:476) line showed significant transcriptome reprogramming rapidly after inoculation, while the other lines showed a delay in this response by about 42 hours. Defense responses are associated with the TIR-NBS, CC-NBS-LRR and NBS-LRR genes, 10 proteins involved in pathogenesis, lipid transfer proteins, endoglucan-1,3-β-glucosidase, glycine-rich cell wall proteins, and genes from reactive path of oxygen. Early responses to 83A:476, including careful suppression of genes associated with photosynthesis, coincided with successful protection during the vegetative growth phase of fungal biology, suggesting that an effector triggers immunity. The Mandeloop reaction is slowed down, as is the overall horizontal drag.
The narrow-leaved lupine (NLL, Lupinus angustifolius L.) is a high protein cereal originating in the western Mediterranean region1,2. It is currently grown as a food crop for animals and humans. It is also considered green manure in crop rotation systems due to nitrogen fixation by symbiotic nitrogen fixing bacteria and overall improvement of soil structure. NLL has undergone a rapid process of domestication in the last century and is still under high breeding pressure3,4,5,6,7,8,9,10,11,12. With the widespread cultivation of NLL, the succession of pathogenic fungi developed new agricultural niches and caused new crop-destroying diseases. The most remarkable for lupin farmers and breeders was the appearance of anthracnose, caused by the pathogenic fungus, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. The most remarkable for lupin farmers and breeders was the appearance of anthracnose, caused by the pathogenic fungus, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Наиболее примечательным для фермеров и селекционеров люпина было появление антракноза, вызванного патогенным грибком Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Most notable to lupine farmers and breeders was the emergence of anthracnose caused by the pathogenic fungus Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.对于羽扇豆农民和饲养者来说，最引人注目的是炭疽病的出现，它是由病原真菌Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13 引起的。对于羽扇豆农民和饲养者来说，最引人注目的是炭疽病的出现，它是由病原真菌Colletotrichum lupini (Bondar)嵵Haired。1 Наиболее поразительным для фермеров и селекционеров люпина является появление антракноза, вызываемого патогенным грибком Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Most striking to lupine farmers and breeders is the emergence of anthracnose caused by the pathogenic fungus Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. The earliest reports of the disease came from Brazil and the United States, with typical symptoms appearing in 1912 and 1929 respectively. However, after about 30 years, the pathogen was designated as Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., телеоморф Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph of Glomerella cingulata (Stoneman) Spauld. & Sacc.，有目的形态的Glomerella cingulata (Stoneman) Spauld。 & Sacc.，有目的形态的Glomerella cingulata (Stoneman) Spauld。 & Sacc., Glomerella cingulata (Stoneman) Spauld в Целенаправленной морфологии. & Sacc., Glomerella cingulata (Stoneman) Spauld in Targeted Morphology. & H. Schrenk,. & H. Schrenk, . and H. Schrenk. & H.施伦克，。 & H.施伦克，。 and H. Schlenk, . Preliminary disease phenotyping done in the mid-20th century showed some resistance in NLL and yellow lupine (L. luteus L.) accessions, but all white lupine (L. albus L.) accessions tested were highly susceptible15,16. Studies have shown that the development of anthracnose is associated with increased precipitation (air humidity) and temperature (in the range of 12-28°C), leading to a violation of resistance at higher temperatures17, 18. In fact, the time required for conidia to germinate and the disease to begin , was four times shorter at 24°C (4 hours) than at 12°C (16 hours) under high humidity conditions19. Thus, the ongoing global warming has led to the spread of anthracnose. However, the disease was observed in France (1982) and Ukraine (1983) as a harbinger of an impending threat, but was apparently ignored by the lupine industry at the time20,21. A few years later, this devastating disease spread throughout the world and also affected major lupine-producing countries such as Australia, Poland and Germany22,23,24. Following an anthracnose outbreak in the mid-1990s, extensive screening resulted in the identification of several resistant donors in NLL19 samples. NLL resistance to anthracnose is controlled by two separate dominant alleles found in different germplasm sources: Lanr1 in cultivar Tanjil and Wonga and AnMan in cultivar. Mandalay 25, 26. These alleles complement the molecular markers that support the selection of resistant germplasm in breeding programs25,26,27,28,29,30. The resistant breeding line 83A:476 carrying the Lanr1 allele was crossed with the susceptible wild line P27255 to obtain an RIL population segregating for anthracnose resistance, which made it possible to assign the Lanr1 locus to chromosome NLL-1131, 32, 33. Alignment of linkage map markers from flanking resistance loci to anthracnose with a genomic framework, NLL revealed the location of all three alleles on the same chromosome (NLL-11), but in different positions29,34,35. However, due to the small number of RILs and the large genetic distance between markers and corresponding alleles, no reliable conclusions can be drawn about their underlying genes. On the other hand, the use of reverse genetics in lupins is difficult due to their very low regeneration potential, which makes genetic manipulation cumbersome37.
The development of domesticated germplasm carrying the desired allele in the homozygous state, such as 83A:476 (Lanr1) and Mandelup (AnMan), has opened the door to studying anthracnose resistance in the face of the presence of opposing combinations of alleles in wild populations. Possibilities of molecular mechanisms. Compare defense responses generated by specific genotypes. This study evaluated the early transcriptome response of NLL to C. lupini vaccination. First, a European NLL germplasm panel containing 215 lines was screened using molecular markers that mark the Lanr1 and AnMan alleles. Anthracnose phenotyping was then performed on 50 NLL lines, previously selected for molecular markers, under controlled conditions. Based on these experiments, four lines differing in anthracnose resistance and Lanr1/AnMan allelic composition were selected for differential defense gene expression profiling using two complementary approaches: high-throughput RNA sequencing and real-time PCR quantification.
Screening of a set of NLL germplasm (N = 215) with markers Lanr1 (Anseq3 and Anseq4) and AnMan (Anseq4) and AnMan (AnManM1) showed that only one line (95726, near Salamanca-b) amplifies the “resistance” allele for all markers , while “Presence of ‘susceptible’ alleles” found the proportion of all markers in 158 (~73.5%) lines. Thirteen lines produced two “resistant” alleles of the Lanr1 marker, and 8 lines produced “resistant” alleles of Lanr1. marker. The “resistance” allele of the AnMan marker (Supplementary Table S1). Two lines were heterozygous for the Anseq3 marker and one heterozygous for the AnManM1 marker. 42 lines (19.5%) carried opposite phases of the Anseq3 and Anseq4 alleles, indicating a high frequency of recombination between these two loci. Anthracnose phenotypes under controlled conditions (Supplementary Table S2) revealed variability in the resistance of the tested genotypes, which was reflected in the severity of anthracnose. Differences in mean scores ranged from 1.8 (moderately resistant) to 6.9 (susceptible) and plant weight differences ranged from 0.62 (susceptible) to 4.45 g (resistant). There was a significant correlation between values observed in two replications of the experiment (0.51 for disease severity scores, P = 0.00017 and 0.61 for plant weight, P < 0.0001) as well as between these two parameters (− 0.59 and − 0.77, P < 0.0001). There was a significant correlation between values ​​observed in two replications of the experiment (0.51 for disease severity scores, P = 0.00017 and 0.61 for plant weight, P < 0.0001) as well as between these two parameters (− 0.59 and − 0.77, P < 0.0001). Выявлена ​​достоверная корреляция между значениями, наблюдаемыми в двух повторностях эксперимента (0,51 для баллов тяжести болезни, P = 0,00017 и 0,61 для массы растения, P < 0,0001), а также между этими двумя параметрами (-0,59 и -0,77, Р < 0,0001) 0,0001). A significant correlation was found between the values ​​observed in two repetitions of the experiment (0.51 for disease severity scores, P = 0.00017 and 0.61 for plant weight, P < 0.0001), as well as between these two parameters (- 0.59 and -0.77, P < 0.0001) 0.0001).在两次重复实验中观察到的值之间存在显着相关性（疾病严重程度评分为0.51，P = 0.00017，植物重量为0.61，P < 0.0001）以及这两个参数之间（- 0.59 和- 0.77，P < 0.0001)。在 两 次 重复 实验 中 观察 的 值 之间 存在 相关性 （疾病 严重 程度 评 分为 分为 分为 0.51 ， p = 0.00017 ， 植物 为 为 0.61 ， p <0.0001） 以及 两 个 参数 之间 （（（（- 0.59 和– 0.59 和– 0.59 和– 0.59 和- 0.77，P < 0.0001)。 Наблюдалась значительная корреляция между значениями, наблюдаемыми в двух повторностях (оценка тяжести заболевания 0,51, P = 0,00017 и масса растения 0,61, P <0,0001), и между этими двумя параметрами (-0,59 и -0,0001) 0,77, P <0,0001. There was a significant correlation between the values ​​observed in duplicate (disease severity score 0.51, P = 0.00017 and plant weight 0.61, P < 0.0001) and between these two parameters (-0.59 and -0 .0001) 0.77, P<0.0001. ). Typical symptoms seen in susceptible plants include kinking and twisting of the stem resembling a “shepherd’s bow” structure, followed by oval lesions with orange/pink sporozoites (Supplementary Fig. 1). Australian accessions carrying the Lanr1 (83A:476 and Tanjil) and AnMan (Mandelup) genes are moderately resistant, 0.0331 and 0.0036). Some lines that also carry “resistant” Lanr1 and/or AnMan alleles show symptoms of the disease.
Interestingly, a few NLL lines lacking any “resistant” marker allele revealed a high level of anthracnose resistance (comparable or higher than for Lanr1 or AnMan genotypes), such as Boregine (P value < 0.0001 for both parameters), Bojar (P value < 0.0001 for score and 0.001 for plant weight) and Population B-549/79b (P value < 0.0001 for score and non-significant for weight). Interestingly, a few NLL lines lacking any “resistant” marker allele revealed a high level of anthracnose resistance (comparable or higher than for Lanr1 or AnMan genotypes), such as Boregine (P value < 0.0001 for both parameters), Bojar (P value < 0.0001 for score and 0.001 for plant weight) and Population B-549/79b (P value < 0.0001 for score and non-significant for weight). Интересно, что несколько линий NLL, лишенных какого-либо «резистентного» маркерного аллеля, показали высокий уровень устойчивости к антракнозу (сопоставимый или более высокий, чем для генотипов Lanr1 или AnMan), таких как Boregine (значение P <0,0001 для обоих параметров), Bojar (значение P < 0,0001 для оценки и 0,001 для массы растения) и популяции B-549/79b (значение P <0,0001 для оценки и незначимо для массы). Interestingly, several NLL lines lacking any ‘resistant’ marker allele showed a high level of resistance to anthracnose (comparable to or higher than for Lanr1 or AnMan genotypes), such as Boregine (P value < 0.0001 for both parameters ), Bojar (P value < 0.0001 for evaluation and 0.001 for plant weight) and population B-549/79b (P value < 0.0001 for evaluation and not significant for weight).有趣的是，一些缺乏任何“抗性”标记等位基因的NLL 系显示出高水平的炭疽病抗性（与Lanr1 或AnMan 基因型相当或更高），例如Boregine（两个参数的P 值< 0.0001）、Bojar（P 值<得分为0.0001，植物重量为0.001）和种群B-549/79b（得分P 值< 0.0001，重量不显着）。 It is interesting that some NLL systems that do not have any “antigenic” markers show high horizontal resistance (equivalent to Lanr1 or AnMan genes or higher), such as Boregine (both parameters P < 0.0001), Bojar (P value < 0.0001, plant weight 0.001) and strain B-549/79b (P value < 0.0001, weight not significant). Интересно, что некоторые линии NLL, лишенные каких-либо маркерных аллелей «резистентности», показали высокие уровни устойчивости к антракнозу (сравнимые или выше, чем у генотипов Lanr1 или AnMan), такие как Boregine (значение P для обоих параметров <0,0001), Bojar (значение P <0,0001, масса растения 0,001) и популяция B-549/79b (оценка P-значение <0,0001, масса незначительна). Interestingly, some NLL lines lacking any ‘resistance’ marker alleles showed high levels of anthracnose resistance (comparable to or higher than Lanr1 or AnMan genotypes), such as Boregine (P value for both parameters <0.0001) , Bojar (P-value < 0.0001, plant weight 0.001) and population B-549/79b (P-value < 0.0001, weight not significant). This phenomenon suggests the possibility of a novel genetic source of resistance, explaining the observed lack of correlation between marker genotypes and disease phenotypes (P values ​​from ~0.42 to ~0.98). Thus, the Kolmogorov-Smirnov test showed that the data on anthracnose resistance were approximately normally distributed for scores (P-values ​​0.25 and 0.11) and plant mass (P-values ​​0.47 and 0.55), suggesting I hypothesize that more alleles than Lanr1 and AnMan are involved.
Based on the results of anthracnose resistance screening, 4 lines were selected for transcriptome analysis: 83A:476, Boregine, Mandelup, and Population 22660. These lines were retested for anthrax resistance in inoculation experiments by RNA sequencing, provided that they were the same as in the previous test. The score values ​​were as follows: Boregin (1.71 ± 1.39), 83A: 476 (2.09 ± 1.38), Mandelup (3.82 ± 1.42) and population 22660 (6.11 ± 1.29 ).
The Illumina NovaSeq 6000 protocol achieved an average of 40.5 Mread pairs per sample (29.7 to 54.4 Mreads) (Supplementary Table S3). Alignment scores in the reference sequence ranged from 75.5% to 88.6%. The average correlation of read count data between experimental variants between biological replicates ranged from 0.812 to 0.997 (mean 0.959). Out of the 35,170 genes analyzed, 2917 revealed no expression, and the other 4785 genes were expressed at negligible level (base mean < 5). Out of the 35,170 genes analyzed, 2917 revealed no expression, and the other 4785 genes were expressed at negligible level (base mean < 5). Из 35 170 проанализированных генов 2917 не проявляли экспрессии, а остальные 4785 генов экспрессировались на незначительном уровне (базовое среднее <5). Of the 35,170 genes analyzed, 2917 showed no expression, and the remaining 4785 genes were expressed at a negligible level (base mean <5).在分析的35,170 个基因中，2917 个没有表达，其他4785 个基因的表达可以忽略不计（基本平均值< 5）。 35,170 Из 35 170 проанализированных генов 2917 не экспрессировались, а остальные 4785 генов имели незначительную экспрессию (базовое среднее значение <5). Of the 35,170 genes analyzed, 2917 were not expressed and the remaining 4785 genes had negligible expression (base mean <5). Thus, the number of genes considered expressed (base mean ≥ 5) during the experiment was 27,468 (78.1%) (Supplementary Table S4).
From the first time point, all NLL lines responded to the inoculation of C. lupini (strain Col-08) by reprogramming the transcriptome (Table 1), however, significant differences were observed between the lines. Thus, the resistance line 83A:476 (carrying the Lanr1 gene) showed significant transcriptome reprogramming at the first time point (6 hpi) with a 31-69-fold increase in the number of isolated up- and down-genes compared to other time points at this time point. In addition, this peak was short-lived, as the expression of only a few genes remained significantly altered at the second time point (12 hpi). Interestingly, Boregine, which also showed a high level of resistance in the graft test, did not undergo such massive transcriptional reprogramming during the experiment. However, the number of differentially expressed genes (DEG) was the same for Boregine and 83A:476 at 12 HPI. Both Mandelup and population 22660 showed DEG peaks at the last time point (48 l/s), indicating a relative delay in defense responses.
Because 83A:476 underwent massive transcriptome reprogramming in response to C. lupini at 6 HPI compared to all other lines, ~91% of the DEGs observed at this time point were lineage-specific (Fig. 1). However, there was some overlap in early responses between study lines, as 68.5%, 50.9%, and 52.6% DEG in Boregine, Mandelup, and population 22660, respectively, overlapped with those found in 83A:476 in certain points in time. However, these DEGs accounted for only a small fraction (0.97–1.70%) of all DEGs currently detected using 83A:476. In addition, 11 DEGs from all lines were coherent at this time (Supplementary Tables S4-S6), including common components of plant defense responses: lipid transfer protein (TanjilG_32225), endoglucan-1,3-β-glucoside enzyme (TanjilG_23384), two stress-inducible proteins like SAM22 (TanjilG_31528 and TanjilG_31531), basic latex protein (TanjilG_32352), and two glycine-rich structural cell wall proteins (TanjilG_19701 and TanjilG_19702). There was also a relatively high overlap in transcriptome responses between 83A:476 and Boregine at 24 HPI (total 16-38% DEG) and between Mandelup and Population 22660 at 48 HPI (total 14-20% DEG).
Venn diagram showing the number of differentially expressed genes (DEG) in narrow-leaved lupine (NLL) lines inoculated with Colletotrichum lupini (strain Col-08 obtained from lupine fields in Wierzhenice, Poland, 1999). The NLL lines analyzed were: 83A:476 (resistant, carrying the Lanr1 allele), Boregine (resistant, genetic background unknown), Mandelup (moderately resistant, carrying the AnMan allele) and population 22660 (very susceptible). The abbreviation hpi stands for hours after vaccination. Zero values ​​have been removed to simplify the graph.
The set of overexpressed genes at 6 hpi was analyzed for the presence of canonical R gene domains (Supplementary Table S7). This study revealed transcriptome induction of classic disease resistance genes with NBS-LRR domains only at 83A:476. This set consisted of one TIR-NBS-LRR gene (tanjilg_05042), five CC-NBS-LRR genes (tanjilg_06165, tanjilg_06162, tanjilg_22773, tanjilg_22640, and tanjilg_16162), and Four NBS-LR, Tanjilg_16162), and Four NBS-LRRE (tanjilg_16162) as well as Four NBS-Lrr (tanjilg_16162) and Four NBS-LRR (TANJILG_16162). All of these genes have canonical domains arranged in conserved sequences. In addition to the NBS-LRR domain genes, several RLL kinases were activated at 6 hpi, namely one in Boregine (TanjilG_19877), two in Mandelup (TanjilG_07141 and TanjilG_19877) and in population 22660 (TanjilG_09014 and TanjilG_10361) and two in 83A 27:476.
Genes with significantly altered expression in response to inoculation with C. lupini (strain Col-08) were subjected to Gene Ontology (GO) enrichment analysis (Supplementary Table S8). The most frequently overrepresented biological process term was “GO:0006952 defense response” which appeared in 6 out of 16 (time × line) combinations with high significance (P value < 0.001) (Fig. 2). The most frequently overrepresented biological process term was “GO:0006952 defense response” which appeared in 6 out of 16 (time × line) combinations with high significance (P value < 0.001) (Fig. 2). Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 защитный ответ», который появлялся в 6 из 16 (время × линия) комбинаций с высокой значимостью (значение P <0,001) (рис. 2). The most frequently overrepresented biological process term was ‘GO:0006952 defense response’, which appeared in 6 of 16 (time × lineage) combinations with high significance (P value < 0.001) (Fig. 2).最常被过度代表的生物过程术语是“GO:0006952 防御反应”，它出现在16 个（时间×线）组合中的6 个中，具有高显着性（P 值< 0.001）（图2）。 The most representative biological process term is “GO:0006952 defense response”, which appears in 6 of the 16 (时间×线) combinations, with high significance (P value < 0.001) (图2) . Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 Defense Response», который появлялся в 6 из 16 комбинаций (время × линия) с высокой значимостью (значение P <0,001) (рис. 2). The most frequently overrepresented biological process term was ‘GO:0006952 Defense Response’, which appeared in 6 out of 16 combinations (time × line) with high significance (P value < 0.001) (Fig. 2). This term was overrepresented at two time points in 83A: 476 and Boregine (6 and 24 hpi) and at one time point in Mandelup and Population 22660 (12 and 6 hpi, respectively). This is an expected result, highlighting the antifungal response of the resistant lines. In addition, 83A:476 responded to C. lupini by rapidly inducing genes related to the oxidative burst represented by the term “GO:0055114 redox process”, indicating a specific defense response, while Boregine revealed specific defense responses, related to the term ‘GO’. :0006950 Stress response”. Population 22660 activated the horizontal resistance response involving secondary metabolites, highlighting the excessive number of terms “GO:0016104 Process of triterpene biosynthesis” and “GO:0006722 Process of triterpene metabolism” (both terms belong to the same set of genes ), taking into account the results of the GO term enrichment analysis, Mandelup reaction stability was between Boregine and Population 22660. In addition, early reaction 83A:476 (6 hpi) and delayed reaction Mandelup and Population 22660 include the term GO:0015979 ‘photosynthesis’ and others related biological processes.
The bioprocess gene ontology terms selected in the annotation of differentially expressed genes during transcriptome responses of narrow-leaved lupine (NLL) inoculated with anthrax lupine (Col-08 strain obtained from lupine fields in Wierzhenice, Poland, in 1999) are greatly exaggerated. The NLL lines analyzed were: 83A:476 (resistant, carrying the homozygous Lanr1 allele), Boregine (resistant, unknown genetic background), Mandelup (moderately resistant, carrying the homozygous AnMan allele) and population 22660 (susceptible).
Because this study aimed to identify genes that contribute to anthracnose resistance, the genes assigned to the terms GO “GO: 0006952 Defensive responses” and “GO: 0055114 Redox processes” were analyzed with cut-offs since baseline means ≥ 30 with at least one line. × point in time combining statistically significant values ​​of log2 (fold change). The number of genes meeting these criteria was 65 for GO:0006952 and 524 for GO:0055114.
83A:476 revealed two DEG peaks annotated with the term GO:0006952, the first at 6 genes per inch (64 genes, up and down regulation) and the second at 24 genes per inch (15 genes, up regulation only). Boregine also showed that GO:0006952 peaked at the same time point, but with less DEG (11 and 8) and preferential activation. Mandeloop showed two peaks of GO:0006952 at 12 and 48 HPI, both carrying 12 genes (the first with activating genes, and the second with only suppressive genes), while the 22660 population at 6 HPI (13 genes) had a greater predominance of the increase peak. regulation. It should be noted that 96.4% of GO:0006952 DEG in these peaks had the same type of response (up or down), indicating a significant overlap in defense responses despite differences in the number of genes involved. The largest group of sequences related to the term GO:0006952 encodes the Starvation Stress-Associated Message Protein 22 (SAM22-like), which belongs to the class 10 pathogenesis-associated protein (PR-10) protein clade and the core protein latex. similar (MLP-like) protein) protein (Fig. 3). The two groups differed in the nature of expression and the direction of the response. The genes encoding SAM22-like proteins showed consistent and significant induction at early time points (6 or 12 hpi) and were generally unresponsive at the end of the experiment (48 hpi), while MLP-like proteins showed coordination at 6 hpi. hpi. 83A:476 and Mandelup at 48 hp/in, almost all other data points were unresponsive. In addition, differences in expression profiles of SAM22-like protein genes followed observed variability in anthracnose resistance, as more resistant lines had more time points significantly inducing these genes than more susceptible genes. Another LlR18A/B-like PR-10 gene showed a very similar expression pattern to the SAM22-like protein gene.
The main components of the biological process term “GO:0006952 Defense Response” and the expression patterns of candidate genes of the Lanr1 and AnMan alleles were identified. The Log2 scale represents log2 values ​​(fold change) between inoculated (Colletotrichum lupini, strain Col-08, obtained from lupine fields, Wizhenica, Poland, 1999) and control (sham-inoculated) plants at the same time point. The following narrowleaf lupine lines were analyzed: 83A:476 (resistant, carrying the homozygous Lanr1 allele), Boregine (resistant, genetic background unknown), Mandelup (moderately resistant, carrying the homozygous AnMan allele), and Population 22660 (susceptible).
In addition, the expression profiles of RNA-seq candidate genes Lanr1 (TanjilG_05042) and AnMan (TanjilG_12861) were evaluated (Fig. 3). The TanjilG_05042 gene showed a significant response (activation) at 83A:476 only at the first time point (6 hpi), while TanjilG_12861 was significant in Mandeloop only at two time points: 6 hpi (down regulation) and 24 hpi (6 hpi). With.). adjustable) ).
The most overexpressed genes in the term GO:0055114 “redox process” were genes encoding cytochrome P450 proteins and peroxidase (Fig. 4). For samples isolated from 83A:476 at 6 HPI, maximum or minimum log2 (fold change) values ​​(for 86.6% of genes) were generally observed between inoculated and control plants, highlighting the high response of this genotype to inoculating sex. 83A:476 showed the most significant GO: 0055114 DEG at 6 hpi (503 genes), while the rest of the lines at 48 hpi (Boregine, 31 genes; Mandelup, 85 genes; and Population 22660, 78 genes)). In most genes of the GO:0055114 family, two types of responses to vaccination (activation and inhibition) were observed. Interestingly, up to 97.6% of the DEGs identified for the term GO: 0055114 in Mandelupe at 48 hp These observations suggest that, despite the significantly smaller scale (i.e., the number of mutated redox genes, 85 versus 503), the pattern of delayed transcriptome responses of mandeloup to anthracnose is similar to the early response of 83A:476. In Boregine and Population 22660, this convergence is lower at 51.6% and 75.6%, respectively.
The patterns of expression of the main components of the term of the biological process “GO:0055114 Redox process” were revealed. The Log2 scale represents log2 values ​​(fold change) between inoculated (Colletotrichum lupini, strain Col-08, obtained from lupine fields, Wizhenica, Poland, 1999) and control (sham-inoculated) plants at the same time point. The following narrowleaf lupine lines were analyzed: 83A:476 (resistant, carrying the homozygous Lanr1 allele), Boregine (resistant, genetic background unknown), Mandelup (moderately resistant, carrying the homozygous AnMan allele), and Population 22660 (susceptible).
83A:476 Transcriptomic responses to inoculation with C. lupini (strain Col-08) also included coordinated silencing of genes attributed to the term GO:0015979 “photosynthesis” and other related biological processes (FIG. 5). This GO:0015979 DEG set contained 105 genes that were significantly repressed at 6 hpi at 83A:476. In this subset, 37 genes were also downregulated in Mandelup at 48 HPI and 35 at the same time point in the 22660 population, including 19 DEGs common to both genotypes. No DEGs related to term GO: 0015979 were significantly activated in any combination (line x time).
The patterns of expression of the main components of the term of the biological process “GO:0015979 Photosynthesis” were revealed. The Log2 scale represents log2 values ​​(fold change) between inoculated (Colletotrichum lupini, strain Col-08, obtained from lupine fields, Wizhenica, Poland, 1999) and control (sham-inoculated) plants at the same time point. The following narrowleaf lupine lines were analyzed: 83A:476 (resistant, carrying the homozygous Lanr1 allele), Boregine (resistant, genetic background unknown), Mandelup (moderately resistant, carrying the homozygous AnMan allele), and Population 22660 (susceptible).
Based on the results of differential expression analysis and presumably involved in defense responses against pathogenic fungi, this set of seven genes was selected for quantification of expression profiles by real-time PCR (Supplementary Table S9).
The putative protein gene TanjilG_10657 was significantly induced in all studied lines and time points compared to control (mimic) plants (Supplementary Tables S10, S11). In addition, the expression profile of TanjilG_10657 showed an increasing trend over the course of the experiment for all lines. Population 22660 showed the highest sensitivity of TanjilG_10657 to inoculation with 114-fold activation and the highest relative expression level (4.4 ± 0.4) at 24 HPI (Fig. 6a). The PR10 LlR18A protein gene TanjilG_27015 also showed activation across all lines and time points, with statistical significance at most data points (Fig. 6b). Similar to TanjilG_10657, the highest relative expression level of TanjilG_27015 was observed in the 22660 inoculated population at 24 HPI (19.5 ± 2.4). The acid endochitinase gene TanjilG_04706 was significantly upregulated in all lines and at all time points except for Boregine 6 hpi (Fig. 6c). It was strongly induced at the first time point (6 HPI) at 83A:476 (by 10.5 times) and moderately increased in other lines (by 6.6-7.5 times). During the experiment, the expression of TanjilG_04706 remained at similar levels in 83A:476 and Boregine, while in Mandelup and Population 22660 it significantly increased, reaching relatively high values ​​(5.9 ± 1.5 and 6.2 ± 1.5, respectively). The endoglucan-1,3-β-glucosidase-like gene TanjilG_23384 showed high activation at the first two time points (6 and 12 hpi) in all lines except population 22660 (Fig. 6d). The highest relative expression levels of TanjilG_23384 were observed at the second time point (12 hpi) in Mandelup (2.7 ± 0.3) and 83A:476 (1.5 ± 0.1). At 24 HPI, TanjilG_23384 expression was relatively low in all studied lines (from 0.04 ± 0.009 to 0.44 ± 0.12).
Expression profiles of selected genes (ag) revealed by quantitative PCR. The numbers 6, 12 and 24 represent hours after vaccination. The LanDExH7 and LanTUB6 genes were used for normalization and LanTUB6 was used for inter-series calibration. Error bars represent the standard deviation based on three biological replicates, each of which is the average of three technical replicates. The statistical significance of differences in the expression levels between the inoculated (Colletotrichum lupini, strain Col-08, obtained in 1999 from the lupin field in Wierzenica, Poland) and control (mock-inoculated) plants are marked above data points (*P value < 0.05, **P value ≤ 0.01, ***P value ≤ 0.001). The statistical significance of differences in the expression levels between the inoculated (Colletotrichum lupini, strain Col-08, obtained in 1999 from the lupin field in Wierzenica, Poland) and control (mock-inoculated) plants are marked above data points (*P value < 0.05, **P value ≤ 0.01, ***P value ≤ 0.001). Статистическая значимость различий в уровнях экспрессии между инокулированными (Colletotrichum lupini, штамм Col-08, получен в 1999 г. с поля люпина в Верженице, Польша) и контрольными (ложно инокулированными) растениями отмечена над точками данных (*значение P < 0,05, **значение P ≤ 0,01, ***значение P ≤ 0,001). Statistically significant differences in expression levels between inoculated (Colletotrichum lupini, strain Col-08, obtained in 1999 from a lupine field in Wierzhenice, Poland) and control (sham-inoculated) plants are noted above the data points (*P value < 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001).接种（Colletotrichum lupini，Col-08株，1999年从波兰Wierzenica的羽扇豆田获得）和对照（模拟接种）植物之间表达水平差异的统计学显着性标记在数据点上方（*P值< 0.05, **P 值≤ 0.01, ***P 值≤ 0.001)。接种 （colletotrichum lupini ， color-08 株 ， 1999 年 波兰 波兰 wierzenica 的 羽扇 获得） 和 对照 （接种 植物 之间 水平 差异 的 统计学 显着性 标记 数据点 上方*p 值 <0.05, **P ≤ 0.01, ***P ≤ 0.001)。 Статистически значимые различия в уровнях экспрессии между инокулированными (Colletotrichum lupini, штамм Col-08, полученный с полей люпина в Верженице, Польша, в 1999 г.) и контрольными (ложно инокулированными) растениями отмечены над точками данных (* значение P < 0,05, ** P-значение ≤ 0,01, ***P-значение ≤ 0,001). Statistically significant differences in expression levels between inoculated (Colletotrichum lupini, strain Col-08, obtained from lupine fields in Verzhenice, Poland, in 1999) and control (sham-inoculated) plants are noted above the data points (*P value < 0.05 , **P-value ≤ 0.01, ***P-value ≤ 0.001). The NLL lines analyzed were: 83A:476 (resistant, carrying the homozygous Lanr1 allele), Mandelup (moderately resistant, carrying the homozygous AnMan allele), Boregine (resistant, unknown genetic background) and population 22660 (susceptible).
The candidate gene TanjilG_05042 at the Lanr1 locus showed a markedly different expression pattern from the profiles obtained from RNA-seq studies (Fig. 6e). Significant activation of this gene was observed in Mandelup and the 22660 population (up to 39.7 and 11.7 times, respectively), resulting in relatively high expression levels (up to 1.4 ± 0.14 and 7.2 ± 1.3, respectively). 83A:476 also revealed some upregulation of the TanjilG_05042 gene (up to 3.8-fold), however, the relative expression levels achieved (0.044 ± 0.002) were more than 30-fold lower than those observed in Mandelup and the 22660 population. analyzed by qPCR showed significant differences in expression levels between genotypes in mock-vaccinated (control) variants, reaching a 58-fold difference between populations 22660 and 83A:476, as well as between populations 22660 and 22660. A two-fold difference was achieved between Boregine and Mandalup.
The candidate gene at the AnMan locus, TanjilG_12861, was activated in response to vaccination in 83A:476 and Mandelup, was neutral in the 22660 population, and was downregulated in Boregine (Fig. 6f). The relative expression of the TanjilG_12861 gene was the highest in inoculated 83A: 476 (0.14±0.01). The 17.4 kDa class I heat shock protein gene TanjilG_05080 HSP17.4 showed lower relative expression levels in all studied strains and time points (Fig. 6g). The highest value was observed at 24 HPI in the 22660 population (0.14 ± 0.02, an eight-fold increase in response to vaccination).
Comparison of gene expression profiles (Fig. 7) revealed a high correlation between TanjilG_10657 and four other genes: TanjilG_27015 (r = 0.89), TanjilG_05080 (r = 0.85), TanjilG_05042 (r = 0.80), and TanjilG_04706 (r = 0.79). Such results may indicate co-regulation of these genes during defense responses. The TanjilG_12861 and TanjilG_23384 genes showed different expression profiles with lower Pearson correlation coefficient values ​​(from 0.08 to 0.43 and -0.19 to 0.28, respectively) compared to other genes.
Correlations between gene expression profiles were detected using quantitative PCR. The following narrowleaf lupine lines were analyzed: 83A:476 (resistant, carrying the homozygous Lanr1 allele), Mandelup (moderately resistant, carrying the homozygous AnMan allele), Boregine (resistant, genetic background unknown), and Population 22660 (susceptible). Three time points were calculated (6, 12 and 24 hours after inoculation), including inoculated (Colletotrichum lupini, strain Col-08, obtained from lupine fields in Wierzhenice, Poland, in 1999) and control (sham-inoculated) plants. The scale shows the value of the Pearson correlation coefficient.
Based on data obtained at 6 horsepower per inch, WGCNA was performed on 9981 DEG identified by comparing inoculated and control plants to focus on early defense responses (Supplementary Table S12). Twenty-two gene modules (clusters) were found with correlated (positive or negative) expression profiles between genotypes and experimental variants. On average, gene expression levels were descending in order 83A:476 > Mandelup > Boregine > Population 22660 (in both variants, however, this trend was stronger in control plants). On average, gene expression levels were descending in order 83A:476 > Mandelup > Boregine > Population 22660 (in both variants, however, this trend was stronger in control plants). В среднем уровни экспрессии генов снижались в порядке 83A:476 > Mandelup > Boregine > Population 22660 (в обоих вариантах, однако, эта тенденция была сильнее у контрольных растений). On average, gene expression levels decreased in the order 83A:476 > Mandelup > Boregine > Population 22660 (in both variants, however, this trend was stronger in control plants).平均而言，基因表达水平按83A:476 > Mandelup > Boregine > Population 22660 的顺序下降（然而，在两种变体中，这种趋势在对照植物中更强）。平均 而 言 ， 基因 水平 按 按 83a: 476> mandelup> boregine> population 22660 的 顺序 下降 （， 在 种 中 ， 这 种 在 在 植物 中 更）。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。 В среднем уровни экспрессии генов снижались в ряду 83A:476 > Mandelup > Boregine > Population 22660 (однако в обоих вариантах эта тенденция была сильнее у контрольных растений). On average, gene expression levels decreased in the series 83A:476 > Mandelup > Boregine > Population 22660 (however, in both variants, this trend was stronger in control plants). Vaccination resulted in upregulation of gene expression, especially in modules 18, 19, 14, 6 and 1 (in descending order of effect), negative regulation (e.g. modules 9 and 20) or with neutral effects (e.g. modules 11, 22, 8 and 13). GO term enrichment analysis (Supplementary Table S13) revealed “GO: 0006952 Protective responses” for the inoculated module (18) with maximum activation, including genes analyzed by qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 and TanjilG_27015), as well as many Inoculate most suppressed photosynthesis modules (9). Module 18 concentrator (Fig. 8) was identified as the TanjilG_26536 gene encoding the PR-10-like LlR18B protein, and module 9 concentrator was identified as the TanjilG_28955 gene encoding the photosystem II PsbQ protein. A candidate anthracnose resistance gene Lanr1, TanjilG_05042, was found in module 22 (Fig. 9) and is associated with the terms “GO:0044260 Cellular macromolecular metabolic processes” and “GO:0006355 Transcriptional regulation, DNA templating” carrying the TanjilG_01212 hub. the gene encodes heat stress transcription factor A-4a (HSFA4a).
Weighted network analysis of gene co-expression of modules with overrepresented biological process terms “GO: 0006952 Defense responses”. Ligation was simplified to highlight the four genes analyzed by qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 and TanjilG_27015).
Weighted network analysis of gene co-expression of a module with an overrepresented biological process term “GO: 0006355: Transcriptional regulation, DNA templating” and carrying a candidate anthracnose resistance gene Lanr1 TanjilG_05042. Ligation was simplified to isolate the TanjilG_05042 gene and the central TanjilG_01212 gene.
Anthracnose resistance screening collected in Australia showed that most of the early released cultivars were susceptible; Kalya, Coromup and Mandelup have been described as moderately resistant, while Wonga, Tanjil and 83A:476 have been described as highly resistant26,27,31. had the same resistance allele, designated Lanr1, and Coromup and Mandelup had a different allele, designated AnMan10, 26, 39, while Kalya passed on a different allele. , Lanr2. Screening for anthracnose resistance in Germany resulted in the identification of a resistant line Bo7212 with a candidate allele other than Lanr1, designated LanrBo36.
Our study revealed a very low frequency (about 6%) of the Lanr1 allele in the tested germplasm. This observation is consistent with the results of screening East European germplasm using the Anseq3 and Anseq4 markers, which showed that the Lanr1 allele is present in only two Belarusian lines. This suggests that the Lanr1 allele is not yet widely used by local breeding programs, unlike in Australia, where it is one of the key alleles for marker-assisted breeding. This may be due to the lower level of resistance provided by the Lanr1 allele in European field conditions compared to the Australian report. In addition, studies of anthracnose in high rainfall areas in Australia have shown that resistance responses mediated by the Lanr1 allele may not be effective in weather conditions that favor growth and rapid development of the pathogen19,42. In fact, in the present study, some symptoms of anthracnose were also observed in genotypes carrying the Lanr1 allele, suggesting that resistance may disappear under optimal conditions for the development of C. lupini. In addition, false positive interpretations of the presence of Anseq3 and Anseq4 markers, which are approximately 1 cM from the Lanr1 locus, are possible 28,30,43 .
Our study showed that 83A:476, carrying the Lanr1 allele, responded to C. lupini inoculation with large-scale transcriptome reprogramming at the first analyzed time point (6 hpi), while in Mandelup, carrying the AnMan allele, transcriptomic responses were observed much later. (from 24 to 48 hp). These temporal variations in defense responses are associated with differences in disease symptoms, highlighting the importance of early pathogen recognition for a successful response to resistance. To infect plant tissue, anthrax spores must go through several developmental stages on the host surface, including germination, cell division, and formation of an appressorium. An appendage is an infective structure that attaches to the surface of the host and facilitates penetration into the host tissues. Thus, spores of C. gloeosporioides in pea extract showed the first division of the nucleus after 75-90 minutes of incubation, the formation of a germ tube after 90-120 minutes, and suppression after 4 hours 45 . Mango C. gloeosporioides showed more than 40% conidial germination after 3 hours of incubation and about 20% formation of appressors after 4 hours. The virulence-associated CAP20 gene of C. gloeosporioides showed transcriptional activity in epiphyte-forming conidia after 3.5 h incubation in avocado surface wax with high concentrations of CAP20 protein after 4 h 46 min. Similarly, the activity of melanin biosynthesis genes in C. trifolii was induced during a 2-hour incubation followed by the formation of an appressorium after 1 hour. Studies of leaf tissues have shown that strawberries inoculated with C. acutatum have first suppression at 8 hpi, while tomatoes inoculated with C. coccodes have first suppression at 4 hpi48,49. largely consistent with the time scale of Colletotrichum spp. infectious process. Rapid defense responses to 83A:476 suggest involvement of plant resistance and effector-triggered immunity (ETI) genes in this line, while Mandelup’s delayed responses support the micro-associated molecular pattern-triggered immunity (MTI) hypothesis 50. Early responses to 83A: 476 and Mandelup. The partial overlap between up- or down-regulated genes in delayed response also supports this concept, as ETI is often considered to be an accelerated and enhanced MTI response that culminates in programmed cell death at the site of infection, known as anaphylactic shock 51,52 .
Most of the genes attributed to the overrepresented term Gene Ontology GO:0006952 “Defense Response” are the 11 homologues of the stress-induced fasting message 22 protein (similar to SAM22) and the seven major latex protein-likes (MLPs). -like proteins 31, 34, 43 and 423 showed sequence similarity. SAM22-like genes showed significant activation that lasted longer, showing increased levels of anthracnose resistance (83A:476 and Boregine). However, MLP-like genes were downregulated only in lines carrying the candidate resistance allele (83A:476/Lanr1 at 6 hpi and Mandelup/AnMan at 24 hpi). It should be noted that all identified SAM22-like homologues originate from a gene cluster spanning approximately 105 kb, while MLP-like genes originate from separate regions of the genome. Coordinated activation of such SAM22-like genes was also found in our previous study of NLL resistance to Diaporthetoxica inoculation, suggesting that they are involved in the horizontal components of the defense response. This conclusion is also supported by reports of a positive response of SAM22-like genes to injury or treatment with salicylic acid, fungal inducers, or hydrogen peroxide.
MLP-like genes have been shown to respond to various abiotic and biotic stresses, including bacterial, viral and pathogenic fungal infections in many plant species55. The directions of response to certain interactions between plants and pathogens ranged from strongly increasing (i.e., during infestation of cotton with Verticillium dahliae) to significantly decreasing (i.e., after infection of apple tree with Alternaria spp.)56,57. Significant downregulation of the MLP-like 423 gene has been observed during avocado defenses to F. niger infection and during infection of the apple tree Botryosphaeria berengeriana f. cn. piricola and Alternaria alternata are apple pathotypes58,59. In addition, apple calli overexpressing the MLP-like 423 gene had lower expression of resistance-associated genes and were more susceptible to fungal infection59. Following Fusarium oxysporum f, the MLP-like 423 gene was also suppressed in resistant common bean germplasm. cn. Bean infection 60.
Other members of the PR-10 family identified in our RNA-seq study were the LlR18A and LlR18B genes in response to upregulation, as well as the upregulated (1 gene) or downregulated (3 genes) gene for the lipid transfer protein DIR1. . In addition, WGCNA highlights the LlR18B gene as a hub in this module, which is highly susceptible to vaccination and carries several protective response genes. The LlR18A and LlR18B genes were induced in yellow lupine leaves in response to pathogenic bacteria, as well as in NLL stems after D. toxica inoculation, while the rice homologue of these genes, RSOsPR10, was rapidly induced by a fungal infection presumably involved in the jasmonic acid signaling pathway53,61, 62. The DIR1 gene encodes nonspecific lipid transport proteins that are required for the onset of systemic acquired resistance (SAR). With the development of protective reactions, the DIR1 protein is transported from the focus of infection through the phloem to induce SAR in distant organs. Interestingly, the TanjilG_02313 DIR1 gene was significantly induced at the first time point in lines 84A:476 and Population 22660, but anthracnose resistance developed successfully only in line 84A:476. This may indicate some subfunctionalization of the DIR1 gene in NLL, since the remaining three homologues responded to inoculation only in the 83A:476 line at 6 hpi, and this response was directed downwards.
In our study, the most common components corresponding to the biological process called “GO:0055114 Redox process” were cytochrome P450 protein, peroxidase, linoleic acid 9S-/13S-lipoxygenase, and 1-aminocyclopropane-1-carboxylic acid oxidase. In addition, our WGCNA defines the HSFA4a homologue as a hub carrying modules such as the Lanr1 resistance gene candidate TanjilG_05042. HSFA4a is a component of the redox-dependent regulation of nuclear transcription in plants.
Cytochrome P450 proteins are oxidoreductases that catalyze NADPH and/or O2-dependent hydroxylation reactions in primary and secondary metabolism, including the metabolism of xenobiotics, as well as hormones, fatty acids, sterols, cell wall components, biopolymers, and the biosynthesis of protective compounds 69. In our In a study, the variability in plant cytochrome P450 function was reduced from -10.6 log2 (fold change) to 5.7 due to a large number of altered homologues (37) and differences in response patterns between specific genes, reflecting an upward revision. . Using only RNA-seq data to elucidate the putative biological function of the NLL genes in such a large protein superfamily would be highly speculative. However, it is worth noting that some cytochrome P450 genes are associated with increased resistance to pathogenic fungi or bacteria, including a contribution to allergic reactions69,70,71.
Class III peroxidases are multifunctional plant enzymes involved in a wide range of metabolic processes during plant growth and development, as well as in response to environmental stresses such as salinity, drought, high light intensity, and pathogen attack72. Peroxidases are involved in the interaction of several plant species with Anthracis, including Stylosanthes humilis and C. gloeosporioides, Lens culinaris and C. truncatum, Phaseolus vulgaris and C. lindemuthianum, Cucumis sativus and C. lagenarium73,74,75,76. The response is very rapid, sometimes even at 4 HPI, before the fungus penetrates the plant tissue73. The peroxidase gene also responded to D. toxica NLL inoculation. In addition to their typical functions to regulate oxidative burst or eliminate oxidative stress, peroxidases can interfere with pathogen growth by creating physical barriers based on cell wall reinforcement during lignification, subunit or cross-linking of specific compounds. This function can be attributed in silico to the TanjilG_03329 gene encoding a putative lignin-forming anion peroxidase that was significantly upregulated in our study in the 83A:476 resistant line at 6 HPI, but not in other strains and time points that did not respond.
9S-/13S-lipoxygenase of linoleic acid is the first step in the oxidative pathway of lipid biosynthesis78. The products of this pathway have multiple functions in plant defense, including cell wall strengthening through the formation of callose and pectin deposits, and regulation of oxidative stress through the production of reactive oxygen species79,80,81,82,83. In the present study, the expression of linoleic acid 9S-/13S-lipoxygenase was altered in all strains, but in the susceptible population 22660, upregulation prevailed at different time points, while in strains carrying resistant Lanr1 and the AnMan allele, it emphasizes the diversification of the oxylipin layer in protective anthrax reactions between these genotypes.
The 1-aminocyclopropane-1-carboxylate oxidase (ACO) homologue was significantly upregulated (9 genes) or downregulated (2 genes) when inoculated with lupine. With two exceptions, all of these responses occurred at 6 hp. at 83A:476. The enzymatic reaction mediated by ACO proteins is the rate-limiting step in ethylene production and is therefore highly regulated84. Ethylene is a plant hormone that plays a variety of roles in regulating plant development and response to abiotic and biotic stress conditions. The induction of ACO transcription and activation of the ethylene signaling pathway are involved in increasing the resistance of rice to the hemibiotrophic fungus oryzae oryzae by regulating the production of reactive oxygen species and phytoalexins. A very similar leaf infection process found between M. oryzae and C. lupini88,89, against the backdrop of a significant upregulation of ACO homologues in the 83A:476 line reported in this study, shifts the possibility of conferring resistance to NLL anthracnose Ethylene a signaling central step in molecular pathways .
In the present study, large-scale suppression of many genes associated with photosynthesis was observed at 6 hpi in 83A:476 and at 48 hpi in Mandeloop and the 22660 population. The extent and progression of these changes is proportional to the level. Anthracnose resistance was observed in this experiment. Recently, strong and early repression of photosynthesis-related transcripts has been reported in several models of plant-pathogen interactions, including pathogenic bacteria and fungi. Haste (from 2 HPI in some interactions) and global suppression of genes associated with photosynthesis in response to infection can trigger plant immunity based on the deployment of reactive oxygen species and their interaction with the salicylic acid pathway to mediate allergic reactions 90,94.
In conclusion, defense response mechanisms proposed for the most resistant lineage (83A:476) include rapid pathogen recognition by the R gene (presumably TIR-NBS-LRR TanjilG_05042) and allergic response-mediated salicylic acid and ethylene signaling, followed by establishment of long-range SAR. action is supported by the DIR-1 protein. It should be noted that the biotrophic period for C. lupini infection is very short (approximately 2 days), followed by necrotic growth95. The transition between these stages may be associated with necrosis and expression of ethylene-inducible proteins that act as triggers for hypersensitivity reactions in host plants. Therefore, the time window for successful capture of C. lupini at the biotrophic stage is very narrow. The reprogramming of genes associated with redox and photosynthesis observed in 83A:476 at 6 hpi is consistent with the progression of fungal hyphae and heralds the development of a successful protective response at the biotrophic stage. The transcriptomic responses of Mandelup and the 22660 population may be too delayed to capture the fungus before switching to necrotic growth, however, Mandelup may be more effective than the 22660 population because the relatively fast regulation of the PR-10 protein promotes horizontal resistance.
ETI, driven by the canonical R gene, appears to be a common mechanism for bean resistance to anthracnose. Thus, in the model legume Medicago truncatula, resistance to anthracnose is conferred by the RCT1 gene, a member of the TIR-NBS-LRR97 plant R gene class. This gene also confers broad-spectrum anthracnose resistance in alfalfa when transferred to susceptible plants. In the common bean (P. vulgaris), more than two dozen anthracnose resistance genes have been identified to date. Some of these genes are found in regions lacking any canonical R genes, however many others are located at the edges of chromosomes carrying the NBS-LRR gene cluster, including TIR-NBS-LRRs99. The genome-wide SSR study also confirmed the association of the NBS-LRR gene with anthracnose resistance in the common bean. The canonical R gene was also found in the genomic region carrying the major anthracnose resistance locus in white lupine 101.
Our work shows that an immediate resistance reaction, activated at an early stage of plant infection (preferably no later than 12 hpi), effectively protects narrow-leaved lupine from anthracnose caused by the pathogenic fungus Collelotrichum lupini. Using high throughput sequencing, we demonstrated differential expression profiles of anthracnose resistance genes in NLL plants mediated by the Lanr1 and AnMan resistance genes. Successful defense involves carefully designing the genes for proteins involved in redox, photosynthesis, and pathogenesis within hours of the plant’s first contact with a pathogen. Similar protective reactions, but delayed in time, are much less effective in protecting plants from diseases. Anthrax resistance mediated by the Lanr1 gene resembles the typical rapid response of the R gene (effector-triggered immunity), while the AnMan gene most likely provides a horizontal response (immunity triggered by a microbe-associated molecular pattern), providing a moderate level of sustainability.
The 215 NLL lines used to screen for anthracnose markers consisted of 74 cultivars, 60 lines obtained by crossing or breeding, 5 mutants, and 76 wild or original germplasms. The lines came from 17 countries, mainly from Poland (58), Spain (47), Germany (27), Australia (26), Russia (19), Belarus (7), Italy (5) and other lines. from 10 countries. The set also includes reference resistant lines: 83A:476, Tanjil, Wonga carrying the Lanr1 allele, and Mandelup carrying the AnMan allele. The lines were obtained from the European Lupine Genetic Resource Database maintained by Poznań Plant Breeding Ltd., Wiatrowo, Poland (Supplementary Table S1).
Plants were grown under controlled conditions (photoperiod 16 hours, temperature 25°C during the day and 18°C ​​at night). Two biological replicates were analyzed. DNA was isolated from three week old leaves using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the protocol. The quality and concentration of the isolated DNA was assessed by spectrophotometric methods (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA). The AnManM1 marker marking the anthracnose resistance gene AnMan (derived from cv. Mandelup) and the markers Anseq3 and Anseq4 flanking the gene Lanr1 (derived from cv. Tanjil) were analyzed 11,26,28. Homozygotes for the resistant allele were scored as “1″, susceptible – as “0″, and heterozygotes – as 0.5.
Based on the results of screening for markers AnManM1, AnSeq3 and AnSeq4 and the availability of seeds for final follow-up experiments, 50 NLL lines were selected for anthracnose resistance phenotyping. The analysis was performed in duplicate in a computer controlled greenhouse with a 14 hour photoperiod with a temperature range of 22°C during the day and 19°C at night. Seeds are scratched (cutting off the seed coat on the opposite side of the embryo with a sharp blade) before sowing to prevent seed dormancy due to the seed coat being too hard and to ensure uniform germination. Plants were grown in pots (11 × 11 × 21 cm) with sterile soil (TS-1 REC 085 Medium Basic, Klasmann-Deilmann Polska, Warsaw, Poland). Inoculation was carried out with the Colletotrichum lupini Col-08 strain, grown in 1999 from the stems of narrow-leaved lupine plants grown in a field in Verzhenitsa, Greater Poland (52° 27′ 42″ N 17° 04′ 05″ E ). Get an area. The isolates were cultured in SNA medium at 20° C. under black light for 21 days to induce sporulation. Four weeks after sowing, when the plants had reached the 4-6 leaf stage, inoculation was carried out by spraying with a suspension of conidia at a concentration of 0.5 x 106 conidia per ml. After inoculation, the plants were kept in the dark for 24 hours at a humidity of about 98% and a temperature of 25°C to facilitate the germination of conidia and the infection process. The plants were then grown under a 14-hour photoperiod at 22°C day/19°C night and 70% humidity. The disease score was made 22 days after inoculation and ranged from 0 (immune) to 9 (very susceptible) depending on the presence or absence of necrotic lesions on stems and leaves. In addition, after scoring, the weight of the plants was measured. Relationships between marker genotypes and disease phenotypes were calculated as point two-sequence correlations (absence of heterozygous markers in the set of lines for analysis of the anthracnose resistance phenotype).