Contribution of the Tobamovirus resistance gene Tm-1 to control of ToBRFV resistance in tomato

Contribution of the Tobamovirus resistance gene Tm-1 to control of ToBRFV resistance in tomato | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Contribution of the Tobamovirus resistance gene Tm-1 to control of ToBRFV resistance in tomato Avner Zinger , Adi Doron-Faigenboim , Dana Gelbart , Ilan Levin , View ORCID Profile Moshe Lapidot doi: https://doi.org/10.1101/2025.01.20.633895 Avner Zinger 1 Institute of Plant Sciences, Agricultural Research Organization – Volcani Institute , 68 HaMaccabim Road, POB 15159, Rishon LeZion 7505101, Israel 2 The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem , POB 12, Rehovot 7610001, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Adi Doron-Faigenboim 1 Institute of Plant Sciences, Agricultural Research Organization – Volcani Institute , 68 HaMaccabim Road, POB 15159, Rishon LeZion 7505101, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dana Gelbart 1 Institute of Plant Sciences, Agricultural Research Organization – Volcani Institute , 68 HaMaccabim Road, POB 15159, Rishon LeZion 7505101, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ilan Levin 1 Institute of Plant Sciences, Agricultural Research Organization – Volcani Institute , 68 HaMaccabim Road, POB 15159, Rishon LeZion 7505101, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Moshe Lapidot 1 Institute of Plant Sciences, Agricultural Research Organization – Volcani Institute , 68 HaMaccabim Road, POB 15159, Rishon LeZion 7505101, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Moshe Lapidot For correspondence: lapidotm{at}volcani.agri.gov.il Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Tomato brown rugose fruit virus (ToBRFV) is a rapidly spreading pathogen that poses a significant threat to tomato production worldwide. We previously identified a locus on tomato chromosome 11 controlling tolerance to the virus. We further established that combining this locus with one that maps to the Tm-1 region on chromosome 2 confers resistance to the virus. Here we sought to determine whether, and how, the Tm-1 gene itself is involved in ToBRFV resistance. Overexpression of Tm-1 in a tolerant genotype significantly reduced viral accumulation, conferring resistance to ToBRFV. On the other hand, overexpression of Tm-1 in a susceptible genotype only delayed symptom appearance. Moreover, effective RNAi-silencing of Tm-1 in the resistant genotype yielded susceptible plants. These findings show that the Tm-1 gene interacts genetically with the locus controlling tolerance on chromosome 11 and that this interaction is critical for achieving effective resistance to ToBRFV. In addition, the symptomatic plants obtained following silencing of Tm-1 in the resistant genotype indicate that tolerance is also dependent on normal expression levels of the recessive tm-1 allele. Introduction Tomato ( Solanum lycopersicum ) is one of the world’s most important crops, serving as a primary food source in human nutrition. Tomatoes are rich in essential metabolites and antioxidants, with related health benefits [ 1 ]. Tomato crops’ adaptability to various climates, including tropical, cool, arid, and desert regions, has contributed to its widespread cultivation. According to the Food and Agriculture Organization of the United Nations (FAO), in 2022, nearly 186 million tons of tomatoes were harvested worldwide, covering approximately 5 million hectares ( https://www.fao.org/faostat/en/#data/QCL ). Plant viruses pose a serious threat to agriculture, reducing both crop yield and quality, with devastating economic consequences. Among the most destructive are the tobamoviruses, which profoundly impact key crops such as tomato, leading to significant challenges for growers. Viruses in the genus Tobamovirus , family Virgaviridae , are characterized by a positive-strand RNA genome that is approximately 6.4 kb in length, and rod-shaped virions [ 2 ]. These viruses are classified according to various criteria: host range, serological properties of their viral particles, amino acid composition of their capsid protein, and nucleotide sequence of their genomes. The Tobamovirus genome harbors four open reading frames: two replication proteins (RdRp) of 126 kDa and their read-through version (183 kDa), a movement protein (MP) of 30 kDa, and a coat protein (CP) of 17.5 kDa [ 2 , 3 ]. The genus comprises about 35 species [ 4 ], with two notable members being tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) [ 5 ]. In contrast to vector-transmitted viruses, tobamoviruses infect plants through microscopic wounds in the tissue and spread via physical contact, which increases their potential for disease transmission. Furthermore, these viruses can persist on seeds and soil, requiring sterilization to ensure virus-free cultivation [ 5 ]. Tobamovirus research has been a focal point in agricultural science. Preventive strategies, such as soil and equipment sterilization, crop rotation, and using clean propagation materials (seeds, cuttings, or tissue culture), have been employed to limit the spread of these viruses [ 5 ]. While helpful, these methods do not offer long-term protection. Genetic resistance has proven to be a more sustainable approach to reducing crop losses [ 6 ], offering relatively efficient management of viral diseases, and providing crop protection without additional labor or material expenses during the growing season [ 7 , 8 ]. Over the past few decades, substantial progress has been achieved in understanding plant-virus interactions. Nearly half of the identified virus-resistance genes in plants exhibit dominant control [ 9 ], while the others display a recessive mode of inheritance, often arising from the absence of crucial host factors necessary for viral replication [ 7 ]. However, continuous evolution of plant viruses calls for new robust and long-lasting resistance strategies [ 7 ]. Advances in breeding have made it possible to enhance genetic resistance to tobamoviruses, particularly ToMV, in cultivated tomatoes. Two essential resistance genes have been identified. One of these, Tomato mosaic virus resistance 1 ( Tm-1 ), was derived from Solanum habrochaites . It is semi-dominant and located on chromosome 2. Tm-1 encodes a ca. 80-kDa protein that binds to and inhibits the replication protein of ToMV [ 3 , 10 , 11 ]. The second gene, Tm-2 , originated from Solanum peruvianum ; it is located on chromosome 9, and has two alleles: Tm-2 and Tm-2 2 [ 10 , 12 ]. Both alleles show dominant inheritance and encode proteins belonging to the CC-NBS-LRR class of resistance proteins [ 13 ]. These proteins disrupt the functionality of the ToMV MP, with Tm-2 2 being more sustainable and thus widely used in tomato breeding programs [ 8 , 14 ]. In 2015, a new Tobamovirus , tomato brown rugose fruit virus (ToBRFV), was identified infecting a commercial tomato hybrid, cv. Candela, in a greenhouse in Jordan. This virus was associated with mild leaf symptoms and distinctive brown rugose symptoms on the fruit [ 15 ]. ToBRFV was also detected in a greenhouse in southern Israel, infecting commercial tomato hybrids carrying the Tm-2 2 resistance gene, indicating the ineffectiveness of this gene at controlling ToBRFV [ 16 , 17 ]. It was shown that changes in the ToBRFV MP sequence, compared to that of ToMV, allow it to evade Tm-2 2 while maintaining its ability to spread [ 18 ]. The impact of ToBRFV was severe, causing dense mosaic patterns on leaves, narrowed foliage, and yellow-spotted fruit, and resulting in significant losses in tomato quantity and quality [ 19 ]. Moreover, ToBRFV can invade the reproductive tissues of tomatoes, including pollen grains; however, although it negatively impacts pollen germination, it is not transmitted through pollen [ 20 ]. Levitzky et al. [ 21 ] also discovered that pollinating insects, particularly bumblebees ( Bombus terrestris ), can spread ToBRFV, further demonstrating its strong potential for transmission. Since its initial identification, ToBRFV has spread globally, affecting major tomato-producing countries such as China, India, Turkey, Italy, Spain, and the United States [ 22 – 26 ]. Since its appearance, efforts have been invested in identifying genetic sources of resistance to ToBRFV. These efforts have resulted in several patents claiming the discovery of resistant or tolerant sources on chromosomes 2, 6, or 9 of the Solanum genome ( https://patentscope.wipo.int/search/en/result.jsf?_vid=P10-LX1RC5-87423 ). In addition, 4 out of 44 tomato accessions screened by Kabas et al. [ 27 ] showed tolerance to ToBRFV. Furthermore, Jewehan et al. [ 28 ] screened 636 Solanum accessions and identified 3 with high resistance to ToBRFV and 26 tolerant accessions. These authors further assessed wild tomato accessions ( S. habrochaites and S. peruvianum ) for their response to infection with ToBRFV. Among the 173 accessions studied, 9 S. habrochaites accessions and 1 S. peruvianum accession demonstrated significant resistance [ 29 ]. These plants remained asymptomatic at 24°C, with no detectable virus in the inoculated leaves. However, upon exposure to 33°C, leaves of the inoculated plants developed mosaic patterns and deformations, indicating susceptibility to the virus at higher temperatures. This research group also reported the discovery of a newly identified mutant isolate of ToBRFV capable of overcoming the previously documented resistance [ 30 ]. We previously identified a single ToBRFV-resistant genotype (VC554) and 29 ToBRFV-tolerant ones [ 31 ]. Among the latter, VC532 was selected for a detailed study, along with VC554. We found that the tolerance trait is controlled by a single recessive gene located on chromosome 11. In contrast, the resistance trait identified in VC554 was controlled by two genes: one located on chromosome 11, allelic to the gene controlling tolerance in VC532, and another locus that mapped to the Tm-1 region on chromosome 2. However, Tm-1 alone did not affect ToBRFV infection [ 31 ]. It therefore remained unclear whether the Tm-1 gene itself participates in this resistance. The main objective of the present study was to further study the role of Tm-1 in conferring resistance to ToBRFV. Based on our previous association studies and considering the established role of Tm-1 in controlling resistance to ToMV [ 3 ], we hypothesized that Tm-1 itself participates in conferring resistance to ToBRFV, potentially through genetic interaction with the locus controlling tolerance on chromosome 11. We employed two approaches to demonstrate the direct involvement of Tm-1 in regulating the resistance trait. In the first, we downregulated the expression level of Tm-1 in the resistant genotype VC554 using RNA interference (RNAi). The second approach involved overexpression of the Tm-1 gene in the tolerant VC532 and susceptible ‘Moneymaker’ genotypes. In addition, to study the architecture of the Tm-1 locus and validate non-synonymous nucleotide changes within the Tm-1 coding sequence (CDS), we used fourth-generation sequencing PacBio technology to sequence the genomes of the three main genotypes participating in this study: the susceptible ‘Moneymaker’ line, the tolerant VC532 line, and the resistant VC554 line. Results The Tm-1 locus is associated with ToBRFV resistance In our previous report [ 31 ], we showed that combining the Tm-1 resistance allele with the locus controlling tolerance on chromosome 11, originating from either the tolerant genotype VC532 or the resistant genotype VC554, yields ToBRFV-resistant plants. On the other hand, combining the susceptible allele tm-1 with those genotypes yielded tolerant plants. To confirm that Tm-1 , in combination with the locus controlling tolerance on chromosome 11, is truly associated with ToBRFV resistance, we inoculated 60 F 3 seedlings segregating at the Tm-1 locus while fixed for the tolerant QTL. Virus-accumulation analyses, individual plant genotyping, and ToBRFV symptom assessment were conducted 30 days post-inoculation (DPI). Results presented in Table 1 show that all of the tested plants displayed a symptomless phenotype [disease severity index (DSI) = 0], as expected due to the effect of the QTL on chromosome 11. Nonetheless, only plants carrying Tm-1 (homozygous Tm-1/Tm-1 and heterozygous Tm-1/tm-1 ) exhibited a significant reduction in virus accumulation [P(F) = 2.54 × 10 -15 , R 2 = 0.69], compared to plants that were homozygous for the susceptible allele ( tm-1/tm-1 ). These results confirmed that Tm-1 is strongly associated with the resistance phenotype in plants carrying the tolerance locus in a homozygous state. However, it remains uncertain whether Tm-1 itself, or a gene or genes that are closely linked to it, contributes to the resistance. To study the direct involvement of Tm-1 in the resistance phenotype, we modulated the expression of the gene using two approaches: RNAi silencing and overexpression. View this table: View inline View popup Download powerpoint Table 1. Tm-1 is associated with the resistance phenotype in plants carrying the tolerance locus. Average disease severity index (DSI) and average ToBRFV level and range in ToBRFV-inoculated control lines and F 3 plants segregating for Tm-1 at 30 DPI Overexpression of Tm-1 in the tolerant VC532 genotype To further validate the interaction between Tm-1 and the locus controlling tolerance on chromosome 11, we overexpressed Tm-1 in the tolerant genotype VC532. This line is homozygous for the locus controlling tolerance on chromosome 11. Of 35 transgenic T 0 plants generated, 11 were randomly selected for Tm-1 expression analysis. Expression levels of Tm-1 in these plants were 4 to 1000 times higher than in the non-transgenic VC532 (data not shown). T 0 plants were self-pollinated to produce T 1 populations segregating for the Tm-1 overexpression transgene ( Tm-1 OE ). Three T 1 populations: TM-127, TM-130, and TM-134, were randomly selected for further analysis and to generate T 2 populations homozygous for the transgene. Analysis of the T 2 plants involved three pairs of populations, each consisting of transgenic and non-transgenic azygous control plants originating from each of three independent T 1 populations. The results of ToBRFV inoculation and Tm-1 expression analysis for the T 2 populations and the control genotypes are summarized in Table 2 . View this table: View inline View popup Table 2. Overexpression of Tm-1 in the tolerant genotype VC532. Average disease severity index (DSI), average ToBRFV level, fold Tm-1 transcription in control plants (A) and in tolerant VC532 T 2 plants overexpressing Tm-1 compared to their azygous control counterparts (B) at 30 DPI Following ToBRFV inoculation, neither of the two control genotypes carrying the locus controlling tolerance on chromosome 11 (VC532 and VC554) showed disease symptoms (DSI = 0). In contrast, the susceptible ‘Moneymaker’ genotype displayed severe disease symptoms at 30 DPI. However, ToBRFV-accumulation levels were high in both the tolerant and susceptible genotypes. As expected, only the resistant genotype VC554, harboring both the locus controlling tolerance on chromosome 11 and the ToMV-resistant gene Tm-1 , had very low virus levels ( Table 2 A). None of the T 2 plants displayed disease symptoms following ToBRFV inoculation (DSI = 0) due to their genetic tolerance background conferred by the locus on chromosome 11. However, a consistent pattern was observed in each of the three T 2 transgenic populations, consisting of elevated Tm-1 expression coupled with a highly significant reduction in virus-accumulation levels compared to their azygous control plants ( Table 2 B). This reduction in virus levels rendered the transgenic plants resistant to the virus and strongly suggests that Tm-1 controls resistance in combination with the locus controlling tolerance on chromosome 11. Overexpression of Tm-1 in a susceptible genotype Our previous findings indicated that the Tm-1 gene alone is ineffective at controlling either tolerance or resistance to ToBRFV [ 31 ]. We therefore overexpressed the gene in susceptible ‘Moneymaker’ plants to validate these results. Introducing Tm-1 under the control of the 35S promoter into ‘Moneymaker’ plants yielded 17 T 0 plants that displayed a diverse range of Tm-1 expression levels, from 0.15 to 18 times higher than those observed in non-transgenic ‘Moneymaker’ plants (data not shown). T 0 plants were self-pollinated to produce T 1 populations segregating for Tm-1 OE . We chose the first three T 1 populations (TM-184, TM-185, and TM-186) to produce seeds and generated three T 2 populations homozygous for the transgene and three azygous counterpart populations. The three pairs of homozygous T 2 populations, alongside their respective control genotypes, were inoculated with ToBRFV and analyzed at 20, 30, and 46 DPI ( Table 3 ). View this table: View inline View popup Tables 3. Overexpression of Tm-1 in the susceptible genotype. Average disease severity index (DSI) at 20, 30, and 46 DPI, average ToBRFV level at 30 DPI, Tm-1 transcription range at 30 DPI, and Tm-1 fold-transcription in control plants (A) and in susceptible T 2 ‘Moneymaker’ plants overexpressing Tm-1 compared to their azygous control counterparts (B) at 30 DPI Tm-1 expression level in the T 2 transgenic plants was, on average, 3- to 14-fold higher than in their non-transgenic azygous counterparts. However, despite this elevated expression, virus-accumulation levels in the transgenic plants were not significantly different from their non-transgenic azygous counterparts. Moreover, they did not differ significantly from the virus level in the control genotypes. Symptom severity, on the other hand, was significantly and consistently lower in the transgenic plants compared to their azygous control counterparts and, at most time points, compared to the control ‘Moneymaker’ and LA2825 ( Tm-1 / Tm-1 genotype in cv. Moneymaker) lines. The relatively higher symptom levels characterizing the transgenic T 2 plants originating from the TM-186 T 1 line can be attributed to their lower-fold average increase in Tm-1 expression levels compared to the other two transgenic lines. Together, these results indicated that overexpression of Tm-1 in a susceptible genotype can yield plants that are at best tolerant to ToBRFV, and they substantiate the hypothesis that a genetic interaction between Tm-1 and the tolerance locus on chromosome 11 is required to yield high ToBRFV resistance. Silencing Tm-1 expression in the resistant VC554 genotype To further understand the role of Tm-1 in ToBRFV resistance, we introduced an RNAi silencing vector ( Tm-1 AS ) into the resistant VC554. The five resultant T 0 transformants displayed a broad spectrum of Tm-1 expression suppression, with reductions ranging from 16% to 95% relative to wild-type VC554 levels (data not shown). Following the self-fertilization of these T 0 plants, we obtained three T 1 progeny populations demonstrating segregation of the Tm-1 AS construct. Three T 1 lines, designated TM-187, TM-188, and TM-189, were selected for in-depth analysis. These lines were subsequently used to develop transgenic and non-transgenic homozygous T 2 lines. To assess the effect of Tm-1 silencing on VC554 resistance, we conducted comparative analyses using three pairs of homozygous T 2 populations. Each pair comprised transgenic (carrying Tm-1 AS ) plants and their non-transgenic (azygous) counterparts derived from a different T 0 progenitor. The results of these analyses are presented in Table 4 . View this table: View inline View popup Table 4. Silencing Tm-1 expression in the resistant VC554 genotype. Average disease severity index (DSI), average ToBRFV level, Tm-1 transcription range, and Tm-1 fold-transcription in Tm-1 -silenced VC554 T 2 plants compared to their azygous control counterparts at 30 DPI The T 2 population originating from the T 1 line TM-187 showed a strong and significant reduction of 83% in Tm-1 expression level in the transgenic population compared to its non-transgenic azygous control. This reduction was associated with significantly higher virus-accumulation levels and disease symptoms in the transgenic plants compared to their azygous counterparts. In contrast, the transgenic T 2 plants originating from the T 1 line TM-188 did not display a reduction Tm-1 expression compared to their azygous non-transgenic controls, indicating ineffective Tm-1 silencing. As expected, this resulted in an insignificant effect on disease symptoms and virus-accumulation levels for the transgenic plants vs. their azygous control counterparts. In the T 2 populations originating from the T 1 line TM-189, a reduction of 50% was observed in Tm-1 expression in the transgenic plants compared to their non-transgenic counterparts. However, this non-significant reduction did not affect virus accumulation or symptom severity in the transgenic plants compared to their respective azygous controls. Whole-genome sequencing of the Tm-1 locus To study the architecture of the Tm-1 locus, validate non-synonymous nucleotide changes within and around the Tm-1 CDS, and map novel genes linked to Tm- 1, we used the fourth-generation Revio TM sequencing technology by PacBio ( https://www.pacb.com/revio ) to sequence the genomes of the three main genotypes examined in this study: the susceptible ‘Moneymaker’ line, the tolerant VC532 line, and the resistant VC554 line. Before the analysis, we generated highly accurate sequencing reads, followed by de novo assembly of chromosome 2, complemented by comprehensive gene-prediction analyses. Our DNA quality-control assessment showed that 90% of the genomic DNA fragments were longer than 10 kb, with 50% exceeding 30 kb, aligning with PacBio’s recommended standards. Using the Revio SMRT cell, we generated 70.7 Gb of data. The HiFi read lengths achieved an N50 of 15.2 kb, with a mean Q-value of 33. All sequences were initially subjected to de novo assembly to reconstruct chromosome 2 for the three genomes (VC554, VC532, and Moneymaker). Gene-prediction analysis, performed using these assemblies, did not reveal any novel gene in the proximal genomic region of Tm-1 compared to the reference genome. However, examining the CDS of Tm-1 across the three genomes tested showed that the resistant genotype VC554 carries two distinct copies of Tm-1 separated by approximately 27 kb. The first copy, referred to as Tm-1 VC554-1st , perfectly matched the nucleotide sequence of the well-documented resistant Tm-1 gene derived from the ToMV-resistant genotype GCR237 ( Tm-1 GCR237 ) [ 3 ]. In contrast, the second copy, referred to as Tm-1 VC554-2nd , shared 97% nucleotide sequence identity with Tm-1 GCR237 , resulting in 95% amino acid sequence identity. Discovery of the two Tm-1 gene copies prompted us to analyze their expression differences (if any) in the VC554 genotype. We conducted a quantitative PCR (qPCR) analysis using two sets of primers: Tm-1_1stq specific to Tm-1 VC554-1st and Tm-1_2ndq specific to Tm-1 VC554-2nd ( Table 5 ). Expression levels of the Tm-1 VC554-2nd gene copy were significantly lower than those of the Tm-1 VC554-1st copy. In fact, the expression level of the second copy was undetectable in some samples (data not shown). View this table: View inline View popup Download powerpoint Table 5. Primers used in this study In contrast to the resistant genotype, sequence analysis of this region in the tolerant VC532 genotype revealed only a single copy of the gene, with a single non-synonymous nucleotide change compared to the reference tm-1 allele sequence identified in the susceptible GCR26 genotype ( tm-1 GCR26 ) by Ishibashi et al. (2007) [ 3 ]. This nucleotide change resulted in a His to Arg change at position 337. Surprisingly, this single-nucleotide polymorphism aligned with the resistance allele Tm-1 GCR237 . As expected, the susceptible ‘Moneymaker’ genotype’s CDS was identical to the susceptible allele sequence of tm-1 GCR26 . Multiple sequence alignment of the Tm-1 CDSs and their corresponding amino acids across the three genomes is shown in the online supplementary material (Figs. S1 and S2). The discovery of two Tm-1 copies in VC554, coupled with the fact that Tm-1 was initially identified in Solanum habrochaites [ 10 ], led us to examine the chromosome 2 sequences of the S. habrochaites reference genome. We found that the S. habrochaites ZY59 reference genome ( https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_027704765.1 ) also carries two Tm-1 copies—7.5 kb and 6.3 kb long—separated by 11.8 kb. Discussion ToBRFV is a rapidly spreading tobamovirus that severely impacts tomato production and fruit quality [ 19 , 32 ]. The most effective, economically viable, and sustainable approach to suppressing ToBRFV’s aggressive replication and inoculation is to identify and introgress resistant genes into commercial breeding lines [ 33 , 34 ]. Our previous work showed that a locus on chromosome 11 controls tolerance to ToBRFV in the VC532 genotype, while the resistance trait in the VC554 genotype results from introducing the locus controlling tolerance on chromosome 11 and the Tm-1 locus on chromosome 2 [ 31 ]. This study aimed to further elucidate the role of Tm-1 in ToBRFV resistance and its interaction with the tolerance locus on chromosome 11. Our findings revealed that the Tm-1 locus, in combination with the tolerance locus on chromosome 11, is essential for conferring ToBRFV resistance. Overexpressing the Tm-1 gene in the tolerant VC532 genotype resulted in a significant reduction in viral accumulation compared to non-transgenic VC532 plants, hence conferring a very high level of resistance to ToBRFV. Conversely, Tm-1 overexpression in the susceptible ‘Moneymaker’ genotype only delayed symptom appearance without significantly affecting viral accumulation, indicating that Tm-1 interacts genetically with the locus controlling tolerance on chromosome 11 to achieve effective resistance against ToBRFV. Our efforts to interfere with Tm-1 expression levels in the resistant VC554 genotype by RNAi revealed varying effects, depending on the extent of expression reduction. A two-fold decrease in Tm-1 expression did not alter resistance. However, a five-fold decrease significantly affected the resistant plants, characterized by elevated virus levels and a substantial increase in symptom severity compared to the non-transgenic azygous VC554 control. Design and analysis of our Tm-1 AS silencing construct were conducted prior to obtaining the PacBio sequencing results. Consequently, during the initial stages of our study, we were unaware of the existence of a second copy of Tm-1 in the VC554 genotype ( Tm-1 VC554-2nd ). We examined whether sequence differences between the two Tm-1 copies in the VC554 genotype could affect the efficiency of Tm-1 silencing. The RNAi construct was designed to target a 303-bp sequence, which exhibited a 10% variation between the two copies. While previous studies have demonstrated that a single RNAi construct can silence multiple homologous genes simultaneously [ 35 , 36 ], we could not confirm whether our construct effectively silenced both Tm-1 copies. Importantly, the Tm-1q primers ( Table 5 ) used for quantifying Tm-1 expression were designed to complement sequences in both Tm-1 copies, ensuring the detection of total Tm-1 transcript abundance. Subsequent qPCR analysis using primers specific to the second Tm-1 copy ( Tm-1_2ndq , Table 5 ) revealed that this copy ( Tm-1 VC554-2nd ) exhibits negligible expression levels in non-transgenic lines (data not shown), suggesting its limited functional relevance. given the extremely low expression of the second copy in non-transgenic lines, our silencing strategy primarily affected the first copy, which appears to be the main functional variant of Tm-1 in this genotype. Given that the VC554 genotype carries the locus controlling tolerance on chromosome 11, we hypothesized that silencing Tm-1 expression would convert the resistant VC554 genotype to a tolerant one, characterized by low symptom severity and high virus accumulation. However, surprisingly, Tm-1 silencing in VC554 rendered the resistant genotype VC554 susceptible, although symptoms levels in the transgenic plants were still less severe than those observed in susceptible plants across all experiments throughout the study. Two potential mechanisms might explain the observed susceptibility phenotype with reduced symptom severity. First, even at high levels of Tm-1 silencing, a certain degree of interaction is maintained, sufficient to reduce symptom severity but not the viral load. Second, these findings suggest that normal expression levels of the recessive tm-1 gene might be essential for achieving complete ToBRFV tolerance. Thus, we propose that ToBRFV resistance requires the interaction of the dominant allele of Tm-1 with the locus on chromosome 11, whereas interaction of the recessive allele tm-1 with the same locus on chromosome 11 induces tolerance to the virus. The limited tolerance observed in transgenic plants originating from TM-187 may reflect the independent action of the chromosome 11 locus, unaffected by the presence of either Tm-1 allele. Our hypothesis that normal expression levels of the recessive tm-1 gene might be essential for obtaining ToBRFV tolerance is supported by a previous study [ 37 ] in which tomato plant resistance to non-host tobamoviruses was suggested to be based on an inhibitory interaction between viral replication proteins and the host cellular protein encoded by tm-1 . Interestingly, our PacBio sequencing analysis revealed that the tm-1 protein in the tolerant VC532 genotype, referred to as tm-1 VC532 , differs by a single amino acid from the susceptible tm-1 GCR26 sequence previously described by Ishibashi et al. [ 3 ], and carried by the ‘Moneymaker’ genotype. Therefore, the possible involvement of this novel tm-1 gene variant in maintaining tolerance in VC532 cannot be dismissed. However, two-way analysis of 220 F 2 plants resulting from a cross between VC532 and ‘Moneymaker’ [ 31 ] showed that tm-1 VC532 has no additive effect on symptom severity [P(F) = 0.74)], and does not interact significantly with the locus on chromosome 11 [P(F) = 0.98)]. As expected, the QTL on chromosome 11 demonstrated a highly significant effect [(P(F)=2.84×10⁻⁷⁵], confirming our previous findings [ 31 ] (results not presented). This lack of effect of tm-1 VC532 on symptom severity is in agreement with our previous results showing that this allele is also ineffective at controlling ToBRFV resistance. Based on these findings, we can conclude that the novel tm-1 allele of the tolerant genotype VC532 ( tm-1 VC532 ) does not contribute to the tolerance mechanism in VC532, and is ineffective at controlling resistance to the virus. A recent study by Kubota et al. [ 38 ] demonstrated that ToBRFV can evolve within Tm-1 -harboring plants, potentially circumventing the Tm-1 effect on resistance. This finding highlights the virus’ adaptability and demonstrates the need for continued studies into the broader impact of viral evolution on resistance mechanisms. In conclusion, our study offers significant insights into the genetic mechanisms underlying resistance and tolerance to ToBRFV in tomatoes. It provides compelling evidence for Tm-1 ’s involvement in ToBRFV resistance, specifically through its interaction with a tolerance locus on chromosome 11. This interaction is essential for achieving durable resistance, characterized by both reduced symptom severity and low virus accumulation. The unexpected susceptibility observed in Tm-1 -silenced VC554 plants suggests a more complex mechanism underlying control of the tolerance trait than previously assumed, leading us to propose that normal expression levels of the recessive tm-1 allele may be essential for achieving complete tolerance to ToBRFV, potentially through interaction with the chromosome 11 locus. Further research is essential to fully elucidate the specific molecular mechanisms underlying the interactions between each of the two Tm-1 gene alleles and the locus on chromosome 11. Understanding these genetic interactions in relation to viral dynamics will be crucial for developing robust and durable ToBRFV-resistant tomato varieties, thereby enhancing crop-protection strategies against this economically significant pathogen. Materials and methods Plant material and resource populations Seeds of tomato accessions were acquired from three sources: the Tomato Genetics Resource Center (TGRC) at the University of California, Davis, USA ( https://tgrc.ucdavis.edu/ ), the laboratory of Professor Dani Zamir at the Hebrew University in Rehovot, Israel, and the Volcani Center seed collection. These genotypes included S. lycopersicum cv. Moneymaker (LA2706) as a susceptible control genotype, a Tm-1/Tm-1 genotype in cv. Moneymaker (LA2825), the tolerant genotype S. lycopersicum var. cerasiforme (VC532), and the resistant genotype, cultivated tomato accession VC554. Development and genotyping of an F 3 population segregating for Tm-1 An F 3 population segregating for the Tm-1 locus while fixed for the locus on chromosome 11 originating from the tolerant VC552 genotype was developed by self-pollination of an F 2 plant. This F 2 plant, homozygous for the VC552-derived chromosome 11 locus and heterozygous for the Tm-1 locus, was obtained from our previously reported allelic test [ 31 ]. Genotyping of the F 3 plants at the Tm-1 locus was conducted using PCR amplification with primers specific to the Tm-1 gene: Tm-1_F and Tm-1_ R ( Table 5 ). The resulting PCR products were digested with Stu I endonuclease to differentiate between genotypes carrying the resistant allele and those carrying the susceptible allele. The digested fragments were visualized by electrophoresis on a 2% agarose gel. Virus maintenance and plant inoculation The ToBRFV isolate (GenBank Acc. No. KXG619418) was maintained on S. lycopersicum cv. Moneymaker plants carrying the ToMV resistance gene Tm-2 2 (LA3310). Plants were maintained in an insect-proof greenhouse, with virus cultures perpetuated through serial mechanical inoculations at 21- to 28-day intervals. For each inoculation, infected leaf tissue was homogenized in 0.01 M phosphate buffer (pH 7.0). The viral homogenate was mechanically applied to carborundum-dusted leaves of test plants. Following inoculation, leaves were rinsed to remove residual carborundum, and plants were transferred to a temperature-controlled greenhouse maintained at 18–25°C under natural photoperiod conditions. Seeds were sown in standardized germination trays (8 × 16 matrix configuration) containing 40 mL of commercial growing medium per cell (Hishtil, Nehalim, Israel). Individual cells served as discrete experimental units for seedling development and subsequent viral challenge. Disease severity scoring ToBRFV-induced symptoms were evaluated 30 DPI and at later times, according to a DSI consisting of: (0) no visible symptoms, with inoculated plants displaying average growth and development, indistinguishable from non-inoculated plants; (1) mild mosaic pattern observed on the apical leaf; (2) distinct mosaic pattern on the apical leaf; (3) pronounced mosaic pattern accompanied by notable elongation or deformation of the apical leaf [ 31 ]. Enzyme-linked immunosorbent assay (ELISA) to evaluate viral levels Indirect ELISA analyses were conducted on plant leaves using specific antibodies against ToBRFV (generously provided by Dr. A. Dombrovsky, ARO, Rishon LeZion, Israel), based on the protocols established by Luria et al. [ 16 ] and Koenig [ 39 ]. Two 1-cm diameter discs were taken from each tested plant’s fourth and fifth leaves. Samples were collected 30 DPI, ground in a coating buffer (Agdia), and incubated for 3 h at 37°C with a 1:5000 dilution of the anti-ToBRFV antiserum. For the subsequent detection step, samples were treated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma, Steinheim, Germany) for 3 additional hours at 37°C. Para-nitrophenylphosphate (Sigma) substrate was used at 0.6 mg mL -1 . The developing color was recorded by a Multiskan FC microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA) at 405 nm and 620 nm. Genomic DNA extraction and PCR Genomic DNA was extracted from individual plants according to Fulton et al. (1995) [ 40 ]. PCR primers were designed using the NCBI primer-blast tool ( https://www.ncbi.nlm.nih.gov/tools/primer-blast ) and are presented in Table 5 . The PCRs were carried out in a 20 µL volume, comprised of 10 µL Hy-taq ready mix by HyLabs ( https://www.hylabs.co.il ), 1 µL of each relevant primer (10 µM), and 8 µL of ultra-pure H 2 O. PCR conditions were: initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 58–60°C for 30 s (depending on primer characteristics), and 72°C for 1 min. The final elongation was at 72°C for 10 min. High-molecular-weight (HMW) DNA extraction, quality assessment, library preparation, and PacBio sequencing HMW plant DNA was extracted from 1.5 g of young leaves using the NucleoBond® HMW DNA kit (Macherey-Nagel, Duren, Germany). Quantity and purity of the HMW DNA were assessed using Qubit Fluorometric Quantification (Thermo Fisher Scientific, Wilmington, NC, USA) and a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific), respectively. DNA-fragment size distribution was analyzed using a FEMTO pulse device (Agilent, Santa Clara, CA, USA). HMW DNA was sent to the DNA Technologies Core facility at UC Davis Genome Center ( https://dnatech.genomecenter.ucdavis.edu/expression-analysis-core/ ). The DNA quality was verified, and sequencing libraries were constructed and subjected to PacBio High Fidelity (HiFi) sequencing using the fourth-generation sequencing technology Revio TM ( https://www.pacb.com/revio ). Bioinformatics analysis of Revio sequence data Raw sequence data generated from the Revio sequencer were assembled by hifiasm (v0.16.1) with default parameters [ 41 ]. Genome-assembly completeness was assessed using Benchmarking Universal Single-Copy Orthologs against single-copy orthologs in the Viridiplantae lineage (BUSCO v5.0.0) [ 42 ], and contiguity was assessed using QUAST (version 5.0.2) [ 43 ]. Repeat elements were detected de novo by RepeatModeler version v1.0.11 and masked by RepeatMasker version 4.0.9_p2 [ 44 ]. Evidence-based gene prediction was performed using Omics Box 2.0.36. The predicted proteins were used as a query term to search the NCBI non-redundant (nr) protein database using the DIAMOND program [ 45 ], and functional annotation was performed in Omics Box 2.0.36. Genome sequences were compared using Mummer software [ 46 ] set at NUCmer on the reference S. lycopersicum cv. Heinz 1706 SL4.0 genome. The cords output of NUCmer was analyzed to extract the homology scaffolds to chromosome 2. Relative transcription level of Tm-1 R elative levels of Tm-1 transcription were determined by real-time qPCR. Total RNA was extracted from 100 mg of young leaf tissue with the NucleoSpin® RNA plant isolation kit (Macherey-Nagel). RNA purity was assessed using a spectrophotometer (Thermo Fisher Scientific). Total RNA (1 µg) was used as the template for first-strand cDNA synthesis with the qScript cDNA Synthesis Kit by Quantabio (Beverly, MA, USA). Tm-1q primers ( Table 5 ) for qPCR were designed using the NCBI primer-blast online tool ( https://www.ncbi.nlm.nih.gov/tools/primer-blast ) to form a 221-bp amplicon. Each qPCR was carried out in a total volume of 12 µL consisting of 6 µL ABsolute Blue qPCR Master Mix with ROX (Thermo Fisher Scientific), 1.2 µL of each primer (0.15 µM), 0.6 µL of ultra-pure H 2 O, and 3 µL of cDNA template. The reactions consisted of: 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. All reactions were conducted in a StepOne Plus™ Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) using a 96-well plate. Data were analyzed using StepOne™ software v 2.3 (Applied Biosystems). Relative expression of the Tm-1 transcript was determined with 18S ribosomal RNA (rRNA) as the reference gene. Amplification was conducted using the 18s_F and 18s _ R primers ( Table 5 ). The calculation was performed using the formula: 2 -(Ct_ Tm-1 -Ct_ 18S ) , where Ct represents the cycle number at which the fluorescence crosses a predefined threshold. The data are presented as fold change in gene expression standardized to the 18S rRNA reference gene and compared to either the non-transgenic or non-transgenic azygous controls. Overexpression of Tm-1 Total cDNA from LA2825 was used in a PCR with Tm-1OESall_F and T m-1OENotI_R primers ( Table 5 ) to develop a Tm-1 CDS clone. This clone, featuring Sall and NotI restriction sequence targets, was inserted into a pBIN vector containing neomycin phosphotransferase II, CaMV 35S promoter, Sal I –Not I sites, and NOS terminator, forming the pBIN Tm-1 -LA2825 expression cassette. Cotyledon cuttings were transformed using Agrobacterium tumefaciens strain GV3103, according to Azari et al. [ 47 ]. T 0 plants overexpressing Tm-1 were generated in VC532 and ‘Moneymaker’ backgrounds and self-pollinated to produce T 1 populations segregating for the Tm-1 OE transgene. Individual T 1 plants were distinguished as either transgenic or non-transgenic using PCR analysis with specific Tm-1OE primers ( Table 5 ). These plants were further self-pollinated to create T 2 populations. Reducing Tm-1 expression level The pHannibal vector [ 48 ], designed to express both sense and antisense fragments of the gene, was constructed through a two-step process. Initially, a 303-bp fragment of the Tm-1 cDNA gene (spanning nucleotides 76 to 378 in the cDNA sequence) was PCR-amplified, utilizing primers Tm-1ASXhoI_F and Tm-1ASEcoRI_R , and introducing XhoI and EcoRI restriction sites, respectively ( Table 5 ). The fragment was inserted into the XhoI and EcoRI sites in the sense-oriented pHannibal region. The identical 303-bp gene fragment was then PCR-amplified using the Tm-1ASXbaI_F and Tm-1ASBamHI_R primers, encompassing an XbaI and BamHI site, respectively ( Table 5 ). This fragment was integrated into the specific XbaI and BamHI sites found in the backward-oriented region of pHannibal, resulting in the creation of pHannibal- Tm-1. To create a binary vector, pHannibal- Tm-1 was inserted under the CaMV 35S promoter and the OCS terminator into the NotI site of the pBIN vector. Transformations were conducted on cotyledon cuttings from VC554 using A. tumefaciens strain GV3101, as previously described [ 47 ]. All T 0 plants were tested by PCR to determine whether they were transgenic using 35S_F and Intron_R specific primers ( Table 5 ). Three T 0 plants were self-pollinated to obtain three T 1 populations segregating for the Tm-1 RNAi construct. We created T 2 populations through self-pollination. For each of the three T 0 plants, we selected 10 transgenic T 1 plants and 2 non-transgenic T 1 plants. These selected plants were then self-pollinated to produce the T 2 generation. Characterization of transgenic plants Integration of the three transformation constructs created in this study was confirmed by PCR using DNA extracted from individual transformed plants as templates. For Tm-1- overexpressing plants, we utilized the transgenic Tm-1 -overexpression primers Tm-1OE ( Table 5 ), each located on a different exon with one intron between them. Consequently, these primers are expected to amplify a genomic fragment of 971 bp. In Tm-1 OE -transgenic plants, these primers will also amplify the incorporated Tm-1 CDS (exons only, without the intron), resulting in an additional 400-bp fragment. To verify the integration of the Tm-1 RNAi ( Tm-1 AS ) construct into VC554 plants, we conducted PCR using the forward primer 35S_F ( Table 5 ), which targets the CaMV promoter, and the reverse primer Intron_R ( Table 5 ), which binds between the sense and antisense regions of the pHannibal vector. This amplification yielded a 623-bp PCR product. Development of homozygous transgenic and non-transgenic T 2 populations We used a progeny-testing approach to obtain homozygous T 2 populations for plants overexpressing Tm-1 in VC532 and in ‘Moneymaker’, as well as Tm-1 silencing in VC554. Transgenic T 0 plants were self-pollinated to produce T 1 populations segregating for the transgene. Individual T 1 plants were identified as either transgenic or non-transgenic using the appropriate primers by PCR and allowed to self-pollinate to obtain T 2 populations. Thirty-two plants of each transgenic and non-transgenic T 2 population were genotyped, identifying and validating homozygous transgenic and non-transgenic T 2 populations for each of the three transgenes. Homozygous T 2 plants were then used for the experiments, where each transgenic and non-transgenic homozygous T 2 population was represented by three biological replicates, each consisting of five plants. Sequence-alignment analysis Nucleotide- and amino acid-sequence alignments of Tm-1 (PacBio) sequenced variants were analyzed and visualized using the Multiple Sequence Alignment (msa) package [ 49 ] and ggmsa package [ 50 ] in R software. Statistical analyses Analyses of variance assessed differences in average virus levels and DSI. All analyses were conducted with the JMP Pro 15 statistical discovery software (SAS Institute Inc., Cary, NC, USA). Differences among means are presented as different superscript letters that represent statistically significant differences between mean values ( P < 0.05), based on the Tukey–Kramer Honestly Significant Difference (HSD) test [ 51 ] or Student’s t -test. Acknowledgments This research was supported by a research grant (award no. IS-5276-20) from BARD (United States – Israel Binational Agricultural Research and Development) to M.L., and by a grant (award no. 20-01-0178) from the Chief Scientist of the Israeli Ministry of Agriculture and Rural Development to M.L. and I.L. The authors wish to thank Aviv Dombrovsky (Volcani Center) for the use of anti-ToBRFV antibodies developed in his laboratory. The presented data are part of the Ph.D. thesis of A.Z., supervised jointly by I.L. and M.L. Footnotes A. Zinger: avnerz{at}volcani.agri.gov.il , A. Doron-Faigenboim: adif{at}volcani.agri.gov.il , D. Gelbart: danag{at}volcani.agri.gov.il , I. Levin: vclevini{at}volcani.agri.gov.il , M. 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Share Contribution of the Tobamovirus resistance gene Tm-1 to control of ToBRFV resistance in tomato Avner Zinger , Adi Doron-Faigenboim , Dana Gelbart , Ilan Levin , Moshe Lapidot bioRxiv 2025.01.20.633895; doi: https://doi.org/10.1101/2025.01.20.633895 Share This Article: Copy Citation Tools Contribution of the Tobamovirus resistance gene Tm-1 to control of ToBRFV resistance in tomato Avner Zinger , Adi Doron-Faigenboim , Dana Gelbart , Ilan Levin , Moshe Lapidot bioRxiv 2025.01.20.633895; doi: https://doi.org/10.1101/2025.01.20.633895 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7622) Biochemistry (17650) Bioengineering (13871) Bioinformatics (41881) Biophysics (21424) Cancer Biology (18566) Cell Biology (25461) Clinical Trials (138) Developmental Biology (13365) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22476) Immunology (17713) Microbiology (40331) Molecular Biology (17148) Neuroscience (88473) Paleontology (666) Pathology (2827) Pharmacology and Toxicology (4816) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)