Fusarium wilt is a destructive soilborne disease of banana caused by Foc, especially by TR4 which is a xylem-invading fungus. TR4 colonizes in the xylem of banana and completes its life cycle . It was evident that xylem sap contained macromolecules, such as proteins, involved in disease-resistance processes [34, 35, 38]. To gain an integrated understanding of the changes of banana xylem sap proteins during TR4 infection, we performed a comparative proteomic analysis of xylem sap in resistant diploid ‘Pahang’ and susceptible triploid ‘Brazilian’ inoculated with TR4 at 14 DPI. A total of 1,036 proteins were detected in xylem sap of both bananas, and were further researched into functions. Some proteins are involved in ‘signal transduction’, ‘environmental adaptation’, ‘biosynthesis of secondary metabolites’ and ‘lipid metabolism’ which are normally regarded as disease-resistance pathway, indicated that xylem sap contained defense-related proteins.
There is no doubt that signal transduction pathways are responsible for induction of plant defense response [41, 42]. Of these pathways, the mitogen-activated protein kinase (MAPK) cascades and plant hormone signals play pivotal roles in plant disease resistance . Once plant perceives the invading pathogen, the activation of MAPKs is one of the earliest signaling events . In the present studies, 10 proteins were related with MAPK signaling pathway-plant (Supplementary Table 1), such as nucleoside diphosphate kinase (NDPK, XP_009384691.1 and XP_009413273.1) inducing MPK3/6 expression through phosphorylation leading to hypersensitive response (HR) cell death in plant response to pathogen attack .
Among the plant hormone signals, salicylic acid (SA) and jasmonic acid (JA) are essential components of plant defense against pathogen . SA and JA antagonize each other . It is generally considered that SA enhances resistance to biotrophs, while JA is effectively against necrotrophs and insects [48, 49]. However, there is exception that SA metabolism activation and signal transduction can improve banana resistance to TR4 . In this study, 2 proteins was associated with SA and JA signals (Supplementary table 1), including pathogenesis-related protein 1 (PR1, XP_009388942.1), a marker for systemic acquired resistance (SAR) from SA signaling pathway ; and coronatine-insensitive protein homolog (COI1, XP_009416210.1), a key regulator for JA-dependent induced systemic resistance (ISR) [48, 52]. Further research is needed to determine whether SA-dependent SAR and JA-dependent ISR were simultaneously activated in banana.
In the natural environment, plants were threatened by various abiotic and biotic stress. Over the evolutionary course during plant-pathogen interaction, plants have developed multi-layered innate immune system to defend against pathogen. The preliminary layer of immune is pathogen-associated molecular pattern (PAMP) perceived by pathogen recognition receptors (PRRs), and induces a series of physiological changes leading to PAMP-triggered immunity (PTI) . These physiological changes include bursts of reactive oxygen species (ROS) and changes in calcium (Ca2+) concentrations [54, 55]. Ca2+ acts as an important second messenger whose concentration is sensed by Ca2+-binding proteins, such as calcium-dependent protein kinase (CDPK, XP_009379843.1) and calcium-binding protein CML7 (CaMCML, XP_009418740.1) detected in the banana xylem sap (Supplementary Table 1), further initiate downstream signaling processes , such as HR and cell wall reinforcement.
Biosynthesis Of Secondary Metabolites
Plant secondary metabolites contribute to all aspects in plant and pathogen interactions . In the biosynthesis of secondary metabolites, phenylpropanoid and flavonoid biosynthesis have been proved to encompass a wide range of constitute and inducible immunity through lignin and phytoalexin synthesis . In the present study, 21 proteins were divided into phenylpropanoid biosynthesis (Supplementary Table 1). The synthetic enzymes of lignin leading to the reinforcement of the cell wall , including phenylalanine ammonia-lyase (PAL, XP_009399473.1), 4-coumarate-CoA ligase (C4L, XP_009384735.1), cinnamoyl-CoA reductase (CCR, XP_009395948.1 and XP_009413954.1), cinnamyl alcohol dehydrogenase (CAD, XP_009397914.1) and peroxidase (POD, XP_009384773.1, XP_009396783.1 and XP_009390142.1) were detected in xylem sap (Supplementary Table 1). In addition, caffeoyl-CoA O-methyltransferase (CCoAOMT, XP_009407208.1) associated with lignin production resulting in quantitative resistance to multiple pathogens , existed also in xylem sap. 13 proteins were assigned to flavonoid biosynthesis, such as chalcone synthase (CHS, XP_009404102.1) as the gatekeeper of flavonoid biosynthesis which can help plant to produce more flavonoids, isoflavonoid-type phytoalexins , the P450 enzyme flavonoid 3',5'-hydroxylase (F3'5'H, XP_009411862.1 and XP_009386727.1) and dihydroflavonol-4-reductase (DFR, XP_009396003.1) as precursors for the production of catechins and pro-anthocyanidins involved in plant resistance .
Lipids and fatty acids involved in lipid metabolism were considered as signal transduction mediators of plant disease resistance [63, 64]. In this study, long chain acyl-CoA synthetases (LACS, XP_009394139.1 and XP_009413949.1) involved in fatty acids metabolism that acting the synthesis of cutin conferred plant resistance to fungal pathogen [65, 66], and phospholipase D α1 (PLDα1, XP_009407292.1, XP_009381115.1 and XP_009408984.1) involved in lipid metabolism which promote phosphatidic acid and ROS affecting plant immunity  were detected in xylem sap (Supplementary Table 1).
Differential Protein Expression Response To Tr4 Infection
To analyze differential protein expression response to TR4 infection, a number of 129 DEPs were identified in 4 possible pairs between resistant and susceptible tested combinations, but only 19 and 11 DEPs were identified in P_dpi vs P_mock and B_dpi vs B_mock, respectively (Supplementary Table 2). It suggested that TR4 did not induce highly dramatic changes in the overall xylem sap proteome. This result was similar to the proteomic analysis of melon phloem sap in response to viral infection . However, these limited DEPs present in phloem sap might also play important roles in banana combatting with TR4.
Hypersensitive-induced response protein 1 (HIR1) may act as regulators of plant immunity by triggering hypersensitive cell death [69, 70]. In the study, HIR1 (XP_018684918.1) decreased in abundance in Pahang but no significant changes occurred in Brazilian under TR4 infection, suggesting Pahang might decrease HIR1 expression to suppress the cell death, and enhanced resistant to TR4 due to Foc usual as hemibiotroph or necrotroph [71, 72].
Ubiquitin involved in the ubiquitination system are key for plant immunity . Ubiquitination is mediated by a three-step enzymatic cascades including activating (E1), conjugating (E2) and ligating (E3) enzymes . E3 has received more attention in research. CaRING1, E3 ubiquitin ligase RING1 gene, played a positive role in pepper (Capsicum annuum) response to microbial pathogens ; whereas a homologous triplet of U-box type E3 ubiquitin ligases acted as negative regulators of PTI in Arabidopsis . In the study, E3 (XP_009408396.1) with a RING zinc-finger domain decreased in abundance only in Pahang response to TR4, however, further studies are needed to prove whether this protein played a negative role in banana response to TR4.
Chalcone isomerase (CHI) is a key enzyme of flavonoid pathway involved in the production of phytoalexin , which plays an important role in plant defense against pathogen. Overexpression of CHI enhanced resistance of soybean (Glycine max) against Phytophthora sojae . In this study, chalcone-flavonone isomerase (also regard as CHI, XP_009384766.1) was increased in abundance in Pahang under TR4 infection, as well as in Pahang mocks compared with Brazilian mocks. It implied that this protein increased resistance against TR4 in banana.
Glycine-rich RNA-binding proteins (GRPs) function as regulators in diverse cellular processes, including response to stress in plants [79, 80]. Over expressing TaRZ1, a wheat (Triticum aestivum) zinc finger-containing GRP, in Arabidopsis thaliana increased resistance against necrotrophic bacteria Pseudomonas syringae . In the present study, GRP (XP_009394303.1) containing an RNA recognition motif (RRM) domain was increased in abundance in Pahang response to TR4, but no significant changes in other pairwise comparisons. It indicated that this protein might play a positive role in banana response against TR4.
Deps Associated Resistance In Pahang
To further explore the associated resistance mechanisms of Pahang, a number of 26 DEPs were identified by comparing TR4 inoculated Pahang with that of Brazilian, and the DEPs in mock inoculated Pahang versus that of Brazilian were excluded. Among which, 7 proteins whose function is unknown, but some proteins are highly associated with resistance to pathogen in plant, such as carboxylesterase and GDSL esterase/lipase (Supplementary Table 2).
Carboxylesterases (CXEs) have been implicated in plant defense. A conserved NbCXE inhibited accumulation of Tobacco mosaic virus (TMV) in Nicotiana benthamiana, which enhanced plant resistance . Constitutive expression of PepEST, a fungus-inducible carboxylesterase in peper (Capsicum annuum) increased resistance against the hemibiotrophic anthracnose fungus (Colletotrichum gloeosporioides) . In the present study, a CXE (XP_009406873.1) was increased abundance in TR4 inoculated Pahang compared with that of Brazilian, but no changes in Pahang mocks compared with that of Brazilian.
GDSL esterase/lipases (GLIP) have been identified in many vascular plants and have been demonstrated that involved in plant defense against pathogens . Overexpressing GLIP1 in Arabidopsis improved resistance against hemibiotrophic and necrotrophic pathogens [85, 86]. In the study, a GLIP (XP_009382515.1) was increased abundance in TR4 inoculated Pahang compared with that of Brazilian, but no changes in other pairs. It further validated our previous transcriptomic study that one GLIP gene was activated by TR4 attack .