Development of Transgenic Brassica Crops against Biotic Stresses Caused by Pathogens and Arthropod Pests

The Brassica genus includes one of the 10 most agronomically and economically important plant groups in the world. Within this group, we can find examples such as broccoli, cabbage, cauliflower, kale, Brussels sprouts, turnip or rapeseed. Their cultivation and postharvest are continually threatened by significant stresses of biotic origin, such as pathogens and pests. In recent years, numerous research groups around the world have developed transgenic lines within the Brassica genus that are capable of defending themselves effectively against these enemies. The present work compiles all the existing studies to date on this matter, focusing in a special way on those of greater relevance in recent years, the choice of the gene of interest and the mechanisms involved in improving plant defenses. Some of the main transgenic lines developed include coding genes for chitinases, glucanases or cry proteins, which show effective results against pathogens such as Alternaria brassicae, Leptosphaeria maculans or Sclerotinia sclerotiorum, or pests such as Lipaphis erysimi or Plutella xylostella.


Introduction
Taxonomically, the Brassica genus belongs to the Brassicaceae family (tribe Brassiceae), which encompasses 338 genera and 3709 species, mostly herbs with annual, biennial or perennial growth habits. They are also known as Cruciferae due to its characteristic flower conformation of four petals arranged in a cross-shape. Brassicaceae species are native to the Irano-Turranian and Mediterranian regions, being distributed in temperate regions. Specifically, Brassica is the most prominent genus in the Brassicaceae family and includes 39 species, many of which are cultivated for their edible roots, leaves, stems, buds, flowers, mustard and oilseeds [1].
Crops belonging to the Brassica genus are among the 10 most economically important vegetable crops in global agriculture and markets. They are mainly cultivated in temperate regions of the

Brassica Transgenesis
Currently, the main strategies for the improvement of Brassica species are molecular breeding and genetic transformation technology. The development of different genetic engineering tools has opened up vast opportunities for the introduction of novel and useful genes of agronomical importance. In this sense, many agronomical important genes have been identified and transferred into Brassica species [10]. Transgenic lines developed so far in the Brassica genus are focused on tolerance to salt stress [11], phytoremediation of heavy metals [12], oil quality improvement, insect resistance, herbicide resistance, development of male-sterile lines and the production of pharmacological and industrial products [10].
The genetic transformation of Brassica has been carried out in almost all the economically important species, but far more work has been conducted with B. napus (rapeseed or canola) than with any of the others. In B. rapa, both oilseed and Chinese cabbage forms have been transformed. B. oleracea transgenic plants include all the major vegetables like cabbage [13], broccoli [14], Brussels sprouts and cauliflower. A few reports dealing with B. nigra, B. juncea and B. carinata have also been conducted.
A fundamental step in the development of transgenic lines is regeneration, which has been achieved in Brassica lines by somatic embryogenesis and organogenesis using different explants. As far as transformation methods are concerned, the Agrobacterium-mediated gene uptake has been most commonly used for Brassica [10]. With regard to Agrobacterium tumefaciens, effective transformations in cotyledonary or hypocotyl explants of Chinese cabbage [15,16], or by floral dip in rapeseed [17] have been reported. In addition, there are perfectly developed protocols for this from seedling explants [18], even having developed an effective transient expression system [19], and also with Agrobacterium rhizogenes [20]. However, nowadays, methods are beginning to be developed with other vector bacteria, such as the rhizospheric bacterium of rapeseed Ensifer adhaerens, which is capable of genetically transforming its original host [21].
An aspect that is always linked to the development of transgenic crops is their safety for the environment, which is based on the capacity of new crops to survive outside the agro-system and the potential exchange of genetic information from these crops to related plants. This is particularly relevant in the case of B. napus because of its known potential for genetic exchange with wild or weedy relatives. In this sense, it has been reported how a glyphosate-resistant rapeseed line, which has been cultivated in Australian fields since 2009, is able to grow naturally outside the agro-system. However, it was also shown how it ends up becoming extinct from the area within 3 years, without being able to become an invasive plant [22]. On the other hand, there are numerous examples of hybridizations between various transgenic crops of the genus Brassica and nearby wild species, such as B. rapa, B. juncea, B. oleracea, B. nigra, Hirschfeldia incana and Raphanus raphanistrum. The recombination of sets of different chromosomes may make not all hybrids viable, but there are reports of hybrids showing good field performance, although they produce fewer seeds than their wild parent [23].

List of Common Pests and Pathogens of Brassica Species
It is estimated that pathogens and pests produced by insects and mites are capable of causing losses in the Brassica crop production of up to 50-60% and in quality, which result in substantial economic losses [24][25][26]. Although biotic stresses may vary depending on the geographical area and the particular crop, there are several of them that are common to many of the different species and varieties grown. Below, we will delve into those Brassica crop pathogens and pests against which effective transgenic lines have been developed.
Regarding bacterial diseases, Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot, is considered to be one of the most important pathogens affecting Brassica vegetables worldwide. There are nine races of Xcc, but races 1 and 4 are considered the most virulent and widespread [27]. Xcc penetrates leaves through hydathodes or wounds. Subsequently, the pathogen travels through the vascular system, thus invading the xylem and colonizing the mesophyll. This causes the appearance of typical symptoms, which include V-shaped chlorosis from the edges of the leaves, necrosis and darkening of leaf veins and the stem vascular tissue [28]. As the disease progresses, wilting and necrosis throughout the plant appear. When the pathogen spreads along the veins and petioles to the plant stems and roots, the vascular tissue within the roots and stems turns black, and the whole plant dies, sometimes accompanied by a secondary soft rot [29]. On the other hand, the most common disease of Brassicaceae is soft rot, mainly caused by highly pectinolytic bacteria Pectobacterium carotovorum (Pc) (formerly Erwinia carotovora). Pc may infect the plant through natural openings or wounds caused by insects, other diseases, or by abiotic factors, such as low temperatures or mechanical damages. For example, symptoms of bacterial soft rot on cabbage include water-soaked lesions, which later become a rotted mass of macerated tissue [30].
As far as fungal diseases are concerned, the most widely distributed pathogen in all Brassica crops are the four species described of genus Alternaria: Alternaria alternata, Alternaria brassicae, Alternaria brassicicola and Alternaria raphani. A. brassicae is a major pathogen of oil-yielding Brassica, while the other three are more common on vegetable crops. All the four Alternaria species cause symptoms on cotyledons at the seedling stage and on leaves, leaf petiole, stem, inflorescence, siliquae and seeds at the adult stage. Quantitative and qualitative losses in the yield of oil seeds and vegetables range from 11 to 100%, depending on the time of infection, prevailing environmental conditions after infection and the strategies used for its control [31].
Sclerotinia stem rot, caused by Sclerotinia sclerotiorum (Ss) fungi, is one of the most devastating diseases for B. napus and B. juncea worldwide, including Australia, China, Europe and North America. In Australia, this disease causes up to 24% yield loss. The pathogen attacks cotyledons and leaves in seedlings and stems and leaves in adult plants, causing some key symptoms like water-soaked lesions, necrotic tissues with fluffy white mycelium and sclerotia inside of stems [32]. In rapeseed, a major constraint in production is blackleg disease, caused by ascomycete fungus Leptosphaeria maculans (Lm). Blackleg has been reported in all canola-growing regions except for China, and causes annual yield losses of 10-20%. In seedlings, the fungus colonizes intercellular spaces, thus causing necrotic cotyledon and leaf lesions before entering a biotrophic phase whilst growing down the petiole and into the stem. At plant maturity, the fungus causes blackening of the stem and cankering, thus restricting the nutrient flow up the plant, and in severe situations, completely killing the plant [33].
Within the group of oomycete pathogens, Albugo candida (Ac) is a biotrophic plant pathogen that causes white blister rust disease in Brassicaceae. Zoospores enter their host through stomata, where they germinate and begin colonization of mesophyll cells. Finally, the oomycete forms zoosporangia that appear as white pustules rupturing the epidermis, hence constituting the visible symptoms of the disease [34]. Additionally, in regard to affecting the aerial part of the plant, we can find the disease known as downy mildew, which is caused by oomycete Hyaloperonospora brassicae (Hb) (formerly Peronospora parasitica). This pathogen causes destructive damages on Brassica, Raphanus and Sinapis species, including on many economically relevant crops, such as broccoli, cabbage, radish, rapeseed, tatsoi and wasabi. In Brassica seedlings, it appears on cotyledons and leaves as a pale green, yellowish growth on leaf undersides, while it causes irregular angular yellow blotches in older plants, which may have dark speckling [35,36].
Turnip mosaic virus (TuMV), family Potyviridae, causes a damaging disease in many kinds of Brassica and other crops worldwide, which results in losses in yield and quality of produce. TuMV is transmitted nonpersistently by more than 40 different aphid species, having a wide natural host range. The disease symptoms often include vein clearing, mosaic, necrosis, plant stunting and plant death, although they depend on the TuMV strain, host plant and environmental conditions [37].
Regarding insect-pests, aphids represent a major constraint to the production of many crops worldwide, especially Brassica crops. Mustard or turnip aphid Lipaphis erysimi pseudobrassicae (Lep) is one of the most destructive pests for Brassica, causing over 50% yield loss. These aphids feed by sucking sap from their host plants, which leads to stunted growth, and they excrete honeydew, which leads to fungal growth (sooty mold). Furthermore, aphids of the L. erysimi group transmit over 13 different viruses, including important viruses of the Brassicaceae, such as TuMV [38].
The Lepidopteran pest, diamondback moth, Plutella xylostella (Px), is the most destructive insect pest for Brassica crops. Larvae feed on host plants' leaves, causing substantial crop losses [39]. Similarly, the large white butterfly, Pieris brassicae (Pb), also known as cabbage white butterfly, can be very destructive, and up to 90% loss in yield of certain Brassica spp. has been reported, especially in developing countries where chemical insecticides are too expensive to be used regularly against this pest [40].

Recent Progress of Transgenic Research in Brassica Species against Pathogens
There has been a large number of transgenic Brassica crops developed against pathogen diseases so far, thanks to a wide variety of different genes from very diverse organisms. Table 1 contains the information on the transgenic lines developed, and indicates information such as the target pathogen or the mechanism involved in resistance. The greatest number of transgenic lines of Brassica crops against fungal pathogens was made by transformation with genes that code for chitinases. Chitinase is an enzyme (a pathogenesis-related protein) that catalyzes the degradation of chitin, which is the main component of the fungal cell wall. In this sense, the most used transformation methodology is from hypocotyls using A. tumefaciens, as it was done in B. juncea, which was transformed with the gene that encodes chitinase C (ChiC) from Streptomyces griseus [88].
Different species belonging to the Trichoderma genus are widely studied and used as biocontrol agents in agriculture due to their ability to parasitize different fungal pathogens, by degrading their cell wall through the release of powerful chitinases [93]. This machinery has been used as a resource in the transformation of Brassica crops, thus obtaining plants that are resistant to Alternaria brassicicola [58,59] and A. brassicae [55] thanks to the endochitinase 42 from Trichoderma harzianum and T. virens. Transgenic lines showed a delayed onset of lesions, as well as a 30-73% reduction in the infected leaf area compared to non-transformed plants. In addition, this chitinolytic effect is increased when a chimeric chitinase produced by the fusion of the Chit42 gene from Trichoderma atroviride and a chitin-binding domain from Serratia marcescens is used, hence causing a great inhibition of fungal growth [61,82].
Other organisms have been used to obtain genes that encode chitinases, such as class I chitinase from tobacco in B. juncea against A. brassicae, which inhibited the fungal colony size by 12-56% over the nontransgenic control [49]. The NIC gene has even been used, coding for a synthetic chitinase, in B. juncea transformation, for which leaf tissue extracts showed considerable resistance and antifungal activity against A. brassicae [60]. We can also mention a double transformation of B. juncea with two genes from barley, class II chitinase and the gene coding for a ribosome inactivating protein (RIP), which inactivates foreign ribosomes in distantly related species and in other eukaryotes, including fungi [51].
Other double transformations with chitinases and other genes have been reported to perform important antifungal activities. Against S. sclerotiorum, transgenic lines of B. napus have been developed that coexpress the Chit42 gene from T. atroviride and a defensin from Raphanus sativus, involved in increasing the fungal membranes permeability [83], or a polygalacturonase inhibiting protein 2 from Phaseolus vulgaris (a protein inhibiting fungal endo-polygalacturonases) [84], showing greater resistance to the disease thanks to a delayed onset and to the restricted size and expansion of lesions when compared to wild type plants. We can also see the chitinase I gene from Paecilomyces javanicus together with a sporamin gene from sweet potato, which is a protein involved in trypsin inhibition [76]. However, in isolation, these genes are also capable of improving the defensive capacity of Brassica crops against different pathogens. For example, the defensive capacity of B. napus against S. sclerotiorum is improved by the transformation with a defensin gene from Orychophragmus violaceus [74] or with a polygalacturonase inhibiting protein 2 gene (PGIP2) from Oryza sativa [85]. PGIP2 genes are not only effective against fungal polygalacturonases, as it has also been observed in the transformation of B. napus with a PGIP2 gene from Proteus vulgaris against Rhizoctonia solani, but they inhibit the disease up to 37% [70]. They are also effective against bacterial polygalacturonases, as in the over-expression of the PGIP2 gene from Chinese cabbage in the same plant, hence exhibiting improved resistance to bacterial soft rot caused by Pc (up to 54%) [45].
Other examples of transformations of Brassica crops with genes that code for defense-related enzymes are found in glucanases, lipases or oxalate oxidases. Since β-1, 3-glucan is a structural polymer present in the fungal cell wall, among fungal resistant genes, glucanases that are potential antifungal agents thanks to their glucan degradation activity are excellent candidates for controlling fungal pathogens development. In this sense, transformation of B. napus with a β-1,3-glucanase from Trichoderma virens showed a stronger inhibition against S. sclerotiorum hyphal growth [80], and the transformation of B. juncea with the class I basic glucanase from tomato showed a restricted number, size and spread of lesions caused by A. brassicae [50]. Similarly, lipases act against the cellular membrane of pathogenic fungi. In B. napus, lipase GDSL1 from Arabidopsis thaliana was characterized as an extracellular GDSL lipase functioning in S. sclerotiorum resistance. By releasing fragments of the fungal membrane, such as phosphatidic acid, GDSL1 activates the defensive responses of the plant through an increase in reactive oxygen species (ROS) and salicylic acid (SA) levels. However, this plant defense response could also be a consequence of the release of phosphatidic acid from the plant plasma membrane, instead of the fungal plasma membrane [86]. During plant infection, S. sclerotiorum secretes oxalic acid, thus acidifying the plant tissue surrounding the site of infection and causing tissue damage, chelating divalent cations and sequestration of calcium that may weaken cell walls, and inhibiting the plant defense route by phenolic compounds. By transforming B. napus plants with a coding gene for enzyme oxalate oxidase from wheat, the breakdown of oxalic acid to CO 2 and H 2 O 2 was achieved, and also a significant reduction in infection caused by the pathogenic fungus [72].
In recent decades, there has been an increased interest in producing transgenic plants that are strongly resistant to a broader spectrum of microbial phytopathogens through the expression of cationic antimicrobial peptides (CAPs). Although CAPs are structurally diverse, most of them fall into two general structure types: α-helical peptides, such as cecropins and magainins, and β-sheet peptides, such as defensins, protegrins, and tachyplesins [94]. In Brassica crops, there are several transformations with genes coding for this type of protein, which have resulted in important inhibitions of pathogen growth and the development of the disease, thanks to a powerful membrane antagonism. Regarding α-helical peptides, we found effective rapeseed transgenic lines against L. maculans [66], the transformation of B. juncea with the gene coding for a cecropin-melittin cationic peptide has been effective against A. brassicae and S. sclerotiorum [53], and also B. napus with a magainin II analogue against S. sclerotiorum [79]. The simultaneous transformation of cauliflower with a cecropin from Antheraea polyphemus and a magainin from Xenopus laevis against Xcc [46] is effective as well. Another group of antimicrobial proteins is found in those known as lipid transfer proteins, which also cause a powerful membrane antagonism against different pathogens. Transgenic lines of B. napus that overexpress these type of genes from Leonurus japonicus and Oryza sativa show greater resistance against S. sclerotiorum, as shown in in vitro experiments with crude leaf extracts from transgenic lines, which significantly inhibit mycelial growth [77,78].
Although Brassica crops have been transformed with genes directly involved in defensive responses against pathogens, they have also been transformed with intermediaries in the signaling of plant responses. Once the plant recognizes the pathogen attack, it activates the plant defense responses through a signaling cascade, where mitogen-activated protein kinases (MAPKs) play a key intermediate role [95]. In Brassica crops, overexpression of MAPKs genes, such as MPK3 and MPK4, increases the resistance of B. juncea and B. napus against A. brassicae and S. sclerotiorum through the activation of the SA-and JA-mediated plant-defenses, respectively [57,73]. WRKY transcription factors are a type of DNA-binding proteins, whose domain is defined by the conserved amino acid sequence WRKYGQK. They are activated by MAPKs and are responsible for activating the gene expression related to the response to SA and JA pathogens [95,96]. Overexpression of WRKY33 in B. napus has shown significant increases in plant resistance against S. sclerotiorum [81]. On the other hand, the non-expressor of pathogenesis-related genes 1 (NPR1) is a master regulator of SA-mediated systemic acquired resistance (SAR), which is a broad-spectrum disease resistance mechanism in plants [95,97]. NPR1 overexpression in B. juncea and B. napus led to an increase in the SA-mediated defensive response and a greater resistance against A. brassicae, E. polygoni and S. sclerotiorum [56,87].
Against viruses, one of the most widely used mechanisms in plant transgenesis is the use of RNA interference that blocks the pathogen molecular machinery. By analyzing a fragment of a 130 bp coat protein, it was possible to develop an efficient hairpin construct against TuMV. The transformation of B. napus with this construction assumes the total absence of viral infection [41].
On the other hand, Brassica crops are not only a sink for genes from other organisms, but they are also used as a source of genes for improving the resistance of other crops against pathogens and pests. In tobacco, the ectopic expression of an annexin from B. juncea confers enhanced resistance against pathogenic oomycete Phytophthora parasitica var. nicotianae, thanks to an increase in message levels for several pathogenesis-related proteins [98]. Additionally, in rice, the expression of B. juncea nonexpressor of pathogenesis-related 1 (BjNPR1) gene, exhibits enhanced resistance against fungi Magnaporthe grisea and Rhizoctonia solani, and bacteria Xanthomonas oryzae pv. oryzae [99]; resistant plants have also been obtained against this bacteria pathogen thanks to transformation with the Brassica rapa cysteine protease 3 (BrCP3) gene [100]. Additionally, in peanut, the co-overexpression of B. juncea NPR1 (BjNPR1) and Trigonella foenum-graecum defensin (Tfgd) provides protection against Aspergillus flavus and Cercospora arachidicola [101].

Recent Progress of Transgenic Research in Brassica Species against Arthropod Pests
Genes used in the transformation of Brassica crops for resistance against pests are less diverse than in the case of defense against pathogens. Although almost all of the transgenic lines developed to date against insects are Bacillus thuringiensis (Bt) crops, there are other examples that share the strategies used against pathogens, which are represented in Table 2. Information such as the target arthropod pest or the mechanism involved in resistance is reflected. As with viruses, the use of interfering RNA has shown good results in controlling herbivorous arthropods. The transformation of Chinese cabbage with complementary fragments of the gene encoding coatomer protein complex subunit 2 (COPB2) derived from mite Tetranichus urticae was achieved. A high mite resistance with nearly a 100% mortality rate was reported. This study demonstrated the effectiveness of the plant-mediated RNAi technique in developing mite-resistant plants [102].   One of the most important direct defense responses in plants against the attack of phytophagous insects is the production of insecticidal peptides or proteins. Among the entomotoxic proteins, we can find the group of carbohydrate-binding proteins or lectins. These proteins, after being ingested by phytophagous insects, are released from disrupted cellular structures and come into contact with carbohydrate structures present in the midgut of insects [146]. In the development of transgenic lines with lectins, there are several examples of the control of aphid Lipaphis erysimi by means of the blockage of the insect gut epithelial membrane and the inhibition of nutrient uptake. In this sense, genes coding for agglutinins (from wheat, garlic or onion) have been the most used in the transformation of B. juncea, reporting a high mortality and significantly reduced fecundity of aphids [103][104][105]. In conjunction with a lentil lectin, the transformation of B. juncea with a chickpea protease inhibitor was achieved, thus reducing aphid survival by 40%. This is because the protease inhibitors of plant origin are basically competitive inhibitors, acting as pseudo-substrates that bind to the active site of respective proteases, thus causing a disruption in assimilation of dietary protein in herbivorous insect pests, which primarily delays their significant growth and development [107].
Sporamins is a widely used group in inhibitor protease transgenesis, and in the transformation of Chinese cabbage [136] and rapeseed against the diamondback moth (Plutella xylostella), in conjunction with chitinase 1 from Paecilomyces javanicus [76]. The cuticle of insect species consists largely of chitin. Therefore, chitinase production has been used as a criterion for selecting potential biocontrol agents against insects. Therefore, because of their ability to interfere with chitin deposition, microbial chitinolytic enzymes have been considered to be important in the biological control of many insects [147]. However, in the transformation of B. napus against P. xylostella, chitinases from Lepidoptera have also been used, such as Manduca sexta, together with the gene encoding for a neurotoxin of contractive paralysis type insect-specific from scorpion Buthus martensii [127].
Bacillus thuringiensis (Bt) is the main microorganism used in the biological control of insect pests. Bt produces a variety of entomotoxic crystalline proteins (Cry) known as δ-endotoxins. When ingested by susceptible insects, crystals dissolve in the insect gut and protoxins are liberated and activated proteolytically to a toxic fragment. This fragment binds to a specific cadherin on the brush border membrane of gut epithelial cells, and inserts into the membrane, thus generating pores. The change in membrane permeability leads to a colloid osmotic lysis of the gut epithelial cells and, ultimately, to insect death. In the last 30 years, numerous transgenic crops expressing Bt insecticidal crystals have been developed. The potential benefits of Bt crops include increased crop yields, a reduction in the use of broad-spectrum insecticide and the associated application costs and energy input, a reduced need for scouting, an improvement of the health conditions of farm workers, and savings in time [148]. In Brassica crops, transgenic-Bt lines of the most cultivated species have been developed against Lepidoptera-pests, such as P. xylostella, Trichoplusia ni, Pieris rapae and Helicoverpa armigera (Table 2), which showed significant resistance. Moreover, it has been proven that the presence of cry genes in the genome of Brassica crops does not modify the synthesis or mode of action of constitutive and herbivore-inducible glucosinolates, hence maintaining an important strategy for these crops against herbivores [130].

Progress on Transgenic Research in Beneficial Biotic Interactions of Brassica Species
Since the first transgenic field, about 25 years ago, different studies have attempted to understand the potential negative effects of this new technology on the environment. A fundamental part is the effect that they can have on different beneficial organisms (soil microorganisms, pollinating insects, etc.) [149].
As far as rhizospheric microorganisms are concerned, studies have only been carried out with the most widely cultivated transgenic Brassica in the world, which is B. napus. Transgenic B. napus harboring the synthetic chitinase (NiC) gene exhibits broad spectrum antifungal resistance, for example against Alternaria brassicicola. Since rhizosphere microorganisms play an important role in element cycling and nutrient transformation, biosafety assessment of NiC containing transgenic plants on the soil ecosystem is a regulatory requirement. By analyzing the rhizosphere enzyme activities and the microbial community structure, it was demonstrated how NiC B. napus lines may not affect rhizospheric microorganisms [150]. The effects produced by herbicide-tolerant transgenic lines must be addressed in a different way, since the application of an herbicide must be analyzed as an indirect effect of the use of a transgenic line. In this sense, by Illumina MiSeq sequencing method, the effect of transgenic glufosinate-tolerant rapeseed and the associated herbicide application on rhizospheric bacterial communities were studied. The results showed that growing glufosinate-resistant transgenic rapeseed line Z7B10 and the application of glufosinate herbicide had no adverse effects on the rhizospheric bacterial community composition [151].
Moreover, several studies have been carried out in soils contaminated with heavy metals in order to determine potential modifications in the beneficial interaction between B. napus and plant growth promoting bacteria (PGPRs). A transgenic rapeseed line that overexpresses the gene for enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase from Pseudomonas putida inoculated with the P. putida bacterium, developed better than its nontransgenic control in nickel-contaminated soils. This shows that the interaction between P. putida and transgenic B. napus leads to greater plant growth and higher heavy metal tissue accumulation [152,153].
In B. napus, the ability of different species of the fungal genus Trichoderma to increase their productivity [154] and tolerance against abiotic stresses such as drought or salinity has been described [155]. In rapeseed plants transformed with the Thkel1 gene from T. harzianum, which codes for a Kelch domain protein, it is pointed out how this protein can modify glucosinolate hydrolysis by binding myrosinase enzymes, hence preventing the formation of toxic compounds such as isothiocyanates. As a consequence, the transgenic rapeseed plants in interaction with T. harzianum significantly increased the levels of fungal root colonization and their productivity. In addition, the presence of this Kelch domain protein reported increases in the β-glucosidase activity, which implies a greater resistance against foliar infection by L. maculans in rapeseed plants [69].
On the other hand, the potential detrimental effects of Bt transgenic lines against non-target arthropods has been widely covered in current reviews [156,157]. The potential detrimental effects of the interaction of transgenic Brassica lines with insects and other beneficial arthropods have been studied in rapeseed and Chinese cabbage. In rapeseed Bt transgenic lines, it has been reported that there could be an exposure to the insecticidal protein of transgenic lines by diurnal pollinators Lepidoptera, as larvae (mainly of Pieridae) through the host and through pollen, and as adults via pollen and nectar. This could lead to a localized loss of biodiversity that requires exhaustive environmental risk assessments [158]. Similarly, the effects of silkworm (Bombyx mori) larvae with pollen from Bt Chinese cabbage have been studied. A decrease in the survival rate and body weight of B. mori larvae fed only with Bt pollen was observed, which was an indication of the negative effect that Bt Chinese cabbage in the field can have on pollinators. However, the authors point out how the reduced interaction of the bumblebee with Chinese cabbage pollen in the field reduces the actual risk to a minimum [159]. Nevertheless, other transgenic lines against pest insects such as rapeseed, which overexpresses pea lectin, have been used to feed bee larvae (Apis mellifera) with their pollen, and it did not show any negative effect on larval mortality, weight or development time [160]. Regarding the effect on herbivore predators that feed on Bt Brassica crops, a study has been carried out with the wolf spider (Pardosa astrigera). After feeding fruit flies (Drosophila melanogaster) with Bt cabbage, it was found that the insecticidal protein had no effect on them, but it did accumulate inside their bodies. The wolf spider was subsequently fed with these fruit flies, not quantifying any negative effect on the predator [161].

Conclusions
The present work compiled the existing studies to date on the interaction between transgenic plants and biotic factors, focusing on those of greater relevance in recent years, the choice of the gene of interest and the mechanisms involved in increasing plant defenses. In the improvement of these crops, numerous transgenic lines have been developed, showing characteristics such as greater tolerance to abiotic stresses, greater phytoremediation capacity or capability of synthesizing and accumulating compounds of industrial interest. In this sense, as a consequence of the importance of pathogens and pests in Brassica crops cultivation (losses of up to 60%), numerous resistant transgenic lines have been developed in recent years.
The transformation of Brassica crops with chitinases has reported numerous lines that are resistant to fungal pathogens in an effective way. Against bacteria and fungi, the transformation with antimicrobial peptides, such as defensins, represents a very effective strategy in the control of these pathogens. On the other hand, modification of plant defense responses through transformation with genes involved in intermediate steps between the recognition of the pathogen and the defensive response has reported significant reductions in the development of diseases in Brassica transgenic crops.
In the development of resistant lines against insect pests, cry genes from Bt are the most widely used, although there are other genes used in Brassica crops. Based on their ability to block intestinal activity, carbohydrate-binding proteins or lectins have been used with significant reductions in the attack of Hemipteran and Lepidopteran pests.
Regarding the interactions of transgenic lines of Brassica crops with beneficial microorganisms from the agro-system, effects of an increasing mutualistic interaction have been reported, without any negative effects being described for the moment. In this sense, it is noteworthy that only B. napus has been used in this type of study, and there is still much to know. On the other hand, the interaction with insects does not fully confirm the absence of negative effects on the natural enemies of pest insects or on pollinators. The development of many other studies is needed, since, until now, only investigations with cabbage and Chinese cabbage exist.

Conflicts of Interest:
The authors declare no conflict of interest.